Medical Biochemistry at a Glance BEN GREENS TEIN aphanns pup ADAM GREENSTEIN Bsc ittons) Medical student b Blackwell Science© 199% by. Blackwell Seience Ld Eira Oe ‘Osnoy Meu, Oxford OX2 OE, 25 ohn Set, Landon WCIN 28 23 Ainslie Place, Edihurgh EHS 6A 238 Main Stet, Cambeie Massachusets 02142. USA, ‘4 University Steet. Carion Victoria MIS). Austlia Other Eira Offices ‘Ameue Blackwell SA Tome de Lille, 7007 Pars France Blackwell Wissenschaft. Verlag Gin ‘Kurfursendam 57 10707 Bern, Germany Zeremenanse 6 AcL1a0 Wien ‘Aust Allrihts served, No par of this publication may be repadced Stored in aretnieval ster, ce ‘ransited in any fom oc by any ‘means elecroni, mechani, hoxacpying recoding or ethers, except as permited bythe UK {Copycight Designs and Patents et 18S, without te pete peeminion of the copyright owner Fist published 1996 Setby Sevite Typeset, Hong Kong Printed and bovind n Great Brian ‘tthe University ross, Cambridge Marsion Book Serves Li PO Box 87 Oxford OX2 007 {Onders Tel 1965 791155 Fay: 01865 791927 Telex: 837515) Non America ‘Blackwel Science, Ine 238 Main tos Cambridge, MA 02142 (Orders el 800-215-1000 1617 876-7000 Fax 617 4925263) Asal ‘Blackwell Science Py Lad 54 University Steet, Carton. Victoria 3053, {Onders Tel 08 9347 0300 ax: 03 9349 3016) ‘catalogue socord or this title valle from the Bish Library ISB 0-86549-980-4 (BSL) ISN 0.86542.626.0 (IE) Library of Congress Caaloing-in-Publication Data Greenstein, Ben, 1981- ‘Medical biochemistry at laced ‘Ben Groemsicin, Adam Greeasten Pam Imcludes bibliographical ferences and inde. ISBN 0-86ss2-0N0-4 1 Biochemisry-Outlne, syllabi ete: 2 Clinical biochemistry Outlines, syllabi, ee 1 Grosrsvin, Adam. Tie IDNEM 1 Biochemisty, QU 4 GRISm 1995] (QPS142.GT6 1986 S74, 19-20 DNLMDLC For Library of Congress 95.8016 cwContents Inieoduction | 1 The eukaryote cell 2 2 Membranes 1 4 3 Membranes I! 6 4 Membranes IIT 8 5) Intracellular receptors and receptor antagonists 10 6 Molecules 112 7 Molecules 14 8 DNA repli 9 DNA repair 18 10. Recombination 20 1 RNA 22 on 16 12. ‘Transcription 1-24 13. Transcription 26 14 Errors of transcription 28 15 Proiein synthesis 1 30 16 Protein synthesis II 32 17. Protein synthesis IIL 34 18 Errors of translation 38 19 Collagen 40 20. Control of gene expression in prokaryotes 42 21 Control of gene expression in eukaryotes 44 22, Mechanisms of transcriptional control 46 23. Growth 48 24 Cancer $0 28. Genetic manipulation I 52 26 Genetic manipulation $4 27 pH and butfers 1 56 pH and butlers I $8 Chemis ‘Chemical reactions IL 62 Enzymes | 64 2 Enzymes 11 66, Enzymes Ill 68 Enzymes IV 70 Digestion: basic principles and cell types 72 Digestion of protein and carbohydrates 74 Digestion and absorption of lipids 76 “The production of energy in the electron transfer chain. 78 Giyeolysis and gluconeogenesis. 80 ‘The citric acid eyele and mitochondrial cariers 82 Glycogen metabolism 84 Lipid metabolism 1 86 Lipid metabolism IL 88 Nucleotide synthesis 90 Breakdown of nucleotides 92 Catabotism of amino acids: 98 Synthesis of amino acids 98 Integration of metabolism 100 Gas transport 102 Haemoglobin 104 Molecular chaperones 106 Abbreviations 108 Glossary 110 Sources 113 Index 115Introduction Medical Biochemistry at a Glance was envisaged, designed and ‘written asa learning aid for undergraduate students, The book consists ‘of aseries of progressively organized, self-contained two-page spreads, in keeping with the general formula of the “AL a Glance’ series. The ‘emphasis has been on the diagrams, with supplementary text, and an attempt has been made to keep the diagrams as simple as possible, given the immense complexity ofthe subject. We hope the reader will find the lagrams rapidly and comfortably accessible, and that they will give the information, literally, ata glance. Although the book has heen called Medical Biochemistry at a Glance it has been written not only for medical students, and we hope that ‘students of Biology, including nurses, will find the book useful, We hhave related biochemical function to disease, bu the emphasis has been fon the basic information and mechanisms. There are branches of Biochemistry which are not covered here, Plant Biochemistzy, for example, isa very important and rapidly growing subject, asi Clinical Biochemistry, which deals in great detail with the biochemistry ‘underlying disease, and with the measurement of clinical biochemical parameters in health and disease. Ii hoped nevertheless tha stadents ‘wishing to specialize in those areas will find this book most useful as an introduction to Biochemistry Biochemistry isa vast subject whose knowledge base expands daily at ‘an almost unbelievable pace, and this book, therefore, modestly hopes to ‘cover only the basic information which underpins the progress that is ‘maul, and which will be required to ensure 2 good examination result for the stodent. The book is not to be thought of as an alternative tothe substantial volumes of Biochemistry which ate available, and which deal with the subject in far more detail ‘We have made every attempt to guarantee the clarity. accuracy and reliability of the information, and have had the contents read by a number ‘of medical students, whose comments and suggestions greatly improved the clarity ofthe diagrams. In alton, the book has been read by several experts, who have contributed immeasurably to the confidence with hich we have produced the book. In particular, we wish to thank Drs Kathleen Rowsell, Richard Gregory. Graham Wallage and Ms Carolyn Watson, who read the entire book and made invaluable comments and suggestions, We ate grateful, also, to Ben Bromilow and to Drs Gavin Brooks, Brian Ellis and Hywel Thomas for reading several of the ‘chapters, It goes without saying, however, that any remaining errors (none we hope) are the responsibility of the authors, and ifthe reader spots any we should lke to kaow, i remains only to thank the editorial staff of Blackwell Science, and particularly Stwart Taylor and Jonathan Rowley for their patience and guidance in bringing this project to a fruitful c Ben Greenstein London, 1995 ‘Adam Greenstein Manchester, 1995Fla, ur 1 The eukaryotic cell INTRODUCTION repations of genes, Algae, protozoa, fungi slime moulds, plans and animals are eukaryotes, Prokaryotes are uniceltlar organisms, including bacteria, whose cells lack @ nucleus ad several other eukaryotic organel Call size In animals cells range in diameter from about 10 to 30 um, Cells are usually microscopic 10 allow diffusion, a process whereby solutes distribute themselves in the available volume, and the rate of diffusion limits cell size. In most cells, no metabolically active region is more ‘than 10-25 jum away from the cel surface, I would take days for amino ‘acids, peptides an sugars to difiuse couple of centimttes, but seconds to travel few micrometees. Mulicellular organisms have overcome this problem by developing a circulation, INTERNAL ARCHITECTURE OF CEL ‘The cell nucleus is bounded by ew membranes: the inner, which defines it, and the outer, whieh is usually continuous with the ‘eytoplasmic endoplasmic reticulum (ER). The nuclear membrane led the perinuclear envelope. The space between jinner and outer membranes is continuous with the lumen of the ER, system is alsoAt points on the nuclear surface, the inner and outer membranes appear to use, creating nuclear pores, which may conduct materials between ‘nucleus and cytoplasm, Most ofthe cell's deoxyribonucleic acid (DNA) ‘occurs in the nucleus, as « DNA-protein complex called chromatin, ‘hich is organized into discrete elongated boxes about 25 nm thick. called chromosomes. The DNA contains the cell's genetic information, lnside the nucleus is the mucleolus, rich in ribonucleic acid (RNA), Within the nucleolus are one or more chromosomes, termed the nucleolar organizer, where ribosomal RNA (rRNA) is formed. ‘The non-ucleolar area of the nucleus is the nucleoplasm, within ‘hich occurs a small family of fibrous proteins termed lamins, which appear to bind DNA to the nuclear membrane, Internal membrane systems ofthe eytoplasm ‘The ER is often the largest internal membrane system of the eukaryotic cell. Rough ER is studded with ribosomes, while smooth ER is no. ‘Smooth ER is the site of synthesis and metabolism of phos Pholipids and fatty acids. Smooth ER contains several enzymes that detoxify carcinogens or pesticides by rendering them water soluble and therefore more easily exereted. This may explain why some cells, such as liver hepatocytes, have more smooth ER than other cell types. Rough ER is present in high abundance in cells that produce peptide hormones, e.g. insulin, and proteins, e.g. antibodies. Ribosomes bound to rough ER produce proteins, forming part of ‘ell and organelle membranes. The FRNA-messenger RNA (mRNA) complex is attached to the ER, and usually passes the elongating Peptide through a pore into the central lumen of the ER, where the ‘chain aggregates prior to transportation elsewhere i the cel, or into the extracellular space The Golgi apparatus is a system of flattened vesicles and smooth ‘membranes which transfers lipid precursors and carbohydrate 10 proteins to form lipoproteins and glycoproteins, respectively, The later process called glycosylation, is necessary for protein transpoet across the plasma membrane. The Golgi also produces much new cellular ‘membrane in the form of vesicles, in which hormones, prohormones and some enzymes are packaged and exported from the cell. They also produce membrane for organelles such as peroxisomes and lysosomes, Lysosomes are organelles with a single membrane, enclosing. acid hydrolase enzymes in an acidic (pH S) environment. The hydrolases degrade polymers such as DNA, RNA and protein into their monomeric units. The lysosomal membrane is impermeable tooth large and small molecules, which are transported through it by specific mediators, Genetic lack of a lysosomal enzyme, B-N-hesosaminidase. which is important in the tumover of a membrane protein, Gy, results in Tay-Sachs disease in which that protein accumulates in developing nerve cells and results in death by age about $ years. Peroxisomes atc small organelles containing enzymes that use oxygen (0,) to oxidize uric acid, p-amino acids and some 2-hydroxyacids, with the production of hydrogen peroxide (HO) HO, is converted in the peroxisome to water (H,O) and O, by catalase. The peroxisome thus protects the cell from HO, powerful ‘oxidant, Peroxisomes also contain enzymes important in lipid ‘metabolism, Peroxisomal enzymes vary from cell to cell, and with ‘changes in cellular conditions. Absence of peroxisomes in brain, kidney liver and skeletal muscle results in arare autosomal recessive disease, Zellweger syndrome, which causes death within 6 months of bith ‘Mitochondria are the energy powerhouses of the cell, They are large, being about 7 jm long and about 0.5-1.0um diameter, and may occupy ‘up to 25% of the cytoplasm. They possess both inner and outer ‘membranes. The outer membrane allows the passage of large molecules, ‘up t9 1OKDa, while the inner membane is less permeable. The inner ‘membrane has many infokdings or eristae, which protrude into the inner space or matrix. Respiratory enzymes protrude from the inner ‘membrane into the matrix, as well a thse which catalyse the production ‘of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). The matrix is filled with the enzymes ‘converting acetyl-coenzyme A (CoA) to carbon dioxide (CO,). Many substances, such as ATP, ADP. citrate and phosphate, which need 10 ‘move into and out ofthe mitochondria, cannot pass passively through ‘the membrane and are transported by permease proteins which form ‘channels for them. Inthe matrix are several copies of a small DNA molecule that codes for several key mitochondrial membrane proteins. Most mitochondrial proteins, however, are produced by cytoplasmic bosomes sing mRNA ‘originating inthe cell nacteus. ‘The eytoskeleton isa network of filaments and microtubules. which maintains cellular morphology, transport, mitosis, meiosis and cell ‘motility, The microtubules are composed of polymers of the protein tubulin, Atleast three mechanochemieal proteins, kinesin dynesin and ch convert chemical into mechanical energy, occur in the myosin, wl cytoskeleton. The cytosol, where many reactions occur, contains soluble constituents. (Note: the ribosome is dealt with more fully on pp. 32-33.)2 Membranes | DT Ed ‘Membrane structure Haro Hyeohie ropa montane pron LIPID MEMBRANES. Functions Membranes: (i) define the shape of an organelle or cell: i) control the ‘exchange of solutes, e.. sodium (Nat), potassium (K°) and chloride (C1) ions between interior and exterior; ii) frm the ste of chemical reactions, 8. oxidative phosphorylation on mitochondrial membeanes (iv) are a site for recognition of chemical messenger and neurotransmitters, whose receptors may be situated on the membrane: (9) have ole in cell-cell recognition; and (vi) facilitate cellular locomotion Architecture A biological membranes range from about $ 10 10-am in thickness, and contain protein and lipid, the ratio of the two varying with the membrane source. Mammalian membranes are especially abundant in ‘phospholipid and cholesterol. The phospholipid bilayer i the common structural unit. Phospholipids are amphipathic, Lc. possessing both hydrophilic and hydrophobie portions in the same molecule. Hydrophobic interactions between the fatty acyl chains of the lipid molecules produce a phospholipid bilayer, 2 sheet o leaflet possess "wo layers of phospholipids whose polar heads face the H,O, sshile the fatty acyl chains form the hydrophobic interior. When phospholipidsa shaken up with H,O, they form spherical micelles, whose fart acyl ‘chains point away from the H.O surface ‘The lipid bilayer is coated on both ses with proteins, and according tothe lu mosaic mextel the lipids themselves and some proteins move round in the plane ofthe bilayer. The membrane proteins serve several functions. They may: (i) transport molecules through the membrane; (i) act as receptors for chemical messengers such as hormones; iit) make possible cell-cell, imeractions through their branching earbohyrate chains, which also ‘make possible the recognition of antigens; and (iv) act as enzymes, Proteins may be integral, being firmly bound to the membrane, or associated, being loosely or reversibly eld to the membrane aad able tobe released by mild treatments, Integral proteins may be anchored, i.e. bonded covalently to the membrane by a link between the carbony terminus ofthe protein and a membrane glycophospholipid (see below), Many integral proteins are insoluble in H,O, and are embeded in the ‘membrane and held to it by three maia forces: (i) ionie interactions with the polar heads (i hydrophobic interactions with the lipid iverior; and (it) specific interactions with cholesterol or other membrane ‘molecules. Most integral proteins span the lipid bilayer and have polar regions at both ends of the protein, MEMBRANE CHEMISTRY Membranes are made up of protein, lipids and varying amounts of elycolipid and glycoprotein (‘glyco implies « sugar moiety) ‘The three main lipids in eukaryotic membranes are cholesterol sphingolipids and phosphoglycerides. Phosphoslycerides are the ‘major membrane lipid components, and the two mest abundant phos= phoglycerides are lecithin (also called phosphatidylehotine) and ‘cephalin (also called phosphatidylethanolamine). Sphingol amphipathic molecules consisting of long-chain fatty acids with an amide linkage, which provides the polar head. Such a compounds termed ceramide. Glycosphingolipids havea sugar moiety either glucose or ids are galactose attached tothe ceramide. Cerebrosides are examples of glyeo- sphingolipids. Galactocerebrosides are cerebrosides found mainly in the central nervous system. Cholesterol is a rigid molecule that intecalates among the phos- pholipids in the membrane Is four- membered hydrophobic steroid ing ieracts with the fatty esl chains of membrane phospholipids. ALST°C, ineukaryotic cells, cholestrol limits the Mudity of the membrane. But, it also prevents the membrane from becoming less fluid at lower temperatures by preventing the chains from binding to each other. Membrane Muidity depends not only on the cholesterol content, but also on temperature and the lipid composition. Fluidity is promoted by shorter, unsaturated fatty acids. There is evidence that fluidity in ‘membranes of certain cells may be influenced by dict, THE ERYTHROCYTE MEMBRANE. ‘The erythrocyte plasma membrane is relatively easily separated from ‘ther constituents. The lipid components are asymmetrically distributed across the membrane, in contrast to their symmerrical distribution in micelles, Forexample, ephalin occurs predominantly on the inner lipid layer. This asymmetry may be maintained by the transverse movement ‘of phospholipids across the membrane, assisted by membrane proteins Using metabolic energy to do so, An uncatalysed ‘lip-lop’ movement ‘of sphingolipid and phosphoglycerides aeross the membrane is slow ‘due tothe tendency for the polar heads not to enter the hydrophobic bilayer, and may take days or weeks ‘The erythrocyte membrane contains an integral glycoprotein, glycophorin, which contains 131 amino acids and spans the membrane, And another ealled band 3, because of is mobility on a polyacrylamide gel after electrophoresis. Band 3, a 900-amino acid protein, may have a role in the facilitated difusion of hydrogen carbonate (HCO, ) and CI across the membrane. It binds the eytosolic peripheral protein ankyrin, which in turn binds the protein spectrin, Spectrin and ankyrin fate members ofthe erythrocyte cytoskeleton,Fig, 3 Membranes II TCU St Ditton Faditaleé tusen (4) Merirene tarspon tar MEMBRANE TRANSPORT The membranc is selectively permeshl a barter to the extracellular environment ii) ensure that essential molecules such as lipids cose and amino acids enter the cell that these stay in the cell and that waste products leave the cell: and ntain ionic gradients across the membrane. Inteacellular ‘organelles may also have selectively permeable membranes. For example, the lysosome membrane maintains a concentration of hydrogen ions (H') 1000 to 100K) times greater than in the cytosol Transport across the membrane may he passive, facilitated or active, ‘Nat K+ ae e | Ap app gy «ile. nce eer |e P ; leat nat 2k ADP acre ego ‘Sympont Ne depundont| reéranetanspot of ‘ADP + Phare phosphate) Nevae-ATPase pump) | transport is the movement of @ molecule or ion down a concentration or electrochemical gradient. It may be by simple diffusion, which is how gases such as O, and CO, and how a simple molecule lke ethanol eross the plasma membrane. In simple difusion, ‘4 smull molecule dissolved in the extracellular uid dissolves in the membrane and then in the cellular fluid. The process is non: specifi and the rate-limiting Facto For entry of the molecule into the its solubility in oil. The rate of merbrane is its hydrophobicity, diffusion through the phospholipid bifayer is proportional to the hydrophobicity. It is also proportional to the concentration gradientFacilitated diffusion i the piel movement of molecules across the membrane, with the help of specific membrane proteins called permeases. The process is specifi, is faster than would be expected from diffusion alone and there isa maximum rate of transpor. ‘Active transport isthe movement of ions ar molecules across the membrane against a concentration gradient, which utilizes energy in the form of ATP hydrolysis to do so. There are three main types of active ion transport: i) the Na’/Kadenosine triphosphatase (ATPase) ‘pump, which transports Na° out and K:* in; (i the calcium ion (Ca) ‘pump (also called the Ca-ATPase pump), which drives Ca" out ofthe cell oF from the eytosol into the sarcoplasmic reticulum: and (ii) the proton (H") pump. lon gradients generated by active transport can be coupled to the active transport of molecules such as certain amino acids and sugars (‘secondary’ active transport Cotransport is the transport of an ion or molecule coupled 10 a cotransported ion. Symport is the simultaneous movement of both in the same direction, while antiport is simultaneous movement in ‘opposite directions. IF anspor is not coupled to a cotransported ion, the process is termed wniport. Cotransport may aceur during facilitated liffusion or during active transport. Glucose can be transported by a symport-facilitated diffusion, while CI- and HCO, ate transported across the erythrocyte membrane by an antiport-facilitated diffusion pump called bind 3, which pumps Cl and HCO, in opposite directions, the direction depending on the prevailing concentration gradient ‘Active transport requires energy generated by ATP hydrolysis to ADP, coupled to the pumping of ions against their concentration gradient: ATP» ADP + Pi, Like fucilitated diffusion, the transport is specific, has saturation kinetics and can be inhibited. An example isthe primary active transport Na‘TK°-ATPase pump. This is an antiporter eneyme system which requires Na’, K* and magnesium ions (Mg), and is ‘present in virtually ll animal cells, with especialy high concentrations in excitable tissues such as nerve and muscle, and in cells actively involved in Na* movement across the plasma membrane. for example the kidney cortex and salivary glands. ‘The ATPase enzyme isan oligomer, composed of two subunits of 110 kDa, and two glycoprotein B-subunits of 5S kDa each, During ATP hydrolysis, the a-subunit is phosphorylated and dephosphorylated fon a specific aspartate residue to form a Braspartamyl phosphate Phosphorylation requires Nav and Me™, but not K°, while dephos- phorylation requires K* but not Na’ or Ma’. The protein complex has bbeen described in two conformations related to energy level, and the ATPase is thus referred to a an E;-E,-type transporter. The ATPase ‘pump is inhibited by the cardiotonie glycosides, including digoxin and ‘ouabain. Ouabain, du wo its high H,O solubility, has been extensively used to characterize the pump. GLUCOSE TRANSPORT Gicosetranspor provides an example of bath aiid difusion and active transport the former utilizing a unipor mechanism athe later 4 sympor mechanism, Glucose can be transported int erythrocytes by facilitated difsion. The Michaels constant (K,) fr glucose uptake into erythrocytes is bout 15 mmol/l (ie. at tis concentration of _slucose, about 0% ofthe available permease molecules wl be bound to plucose molccues). Since the concentration of glucose inhuman blood is around 4-6 mmol, lucose uptake into erythrocytes wil our at virally maximum ates. The permease is specifi, since the -somer Of glucose is not significantly transported ito the erythrocyte. trealactose and -mannose are transported, but higher concentrations ane required taf saturate the transport system. Onc nse the el lucose is posphonated and can no longs lev the cel The permease for glucose is also termed a p-herone permess, It isan integral membrane protein of moleclar weight 45 kDa Glucoxe can alsa be anspor bya Na-dependent symprt system found in plasma membranes oftsue,incing the kidney tubule and intestinal epithelium, One molecule of glucose is moved against is concentration gradient, and one jon of Na is moved down is concentration gradi by facilitated difasion. But, the system is imately diveny the Ne'K*~ATPase pump, The sympa is thereore 2 secondary active transport system. Amino acids are similarly transported THE CAY PUMP. ‘The Ca"*pump is an EE, -type active transport pump. I isan integral ‘membrane protein, phosphorylated on an aspartamyl residue during Ca" transport, Two Ci ions are transported for each molecule of ATP. hydrolysed. In eukaryotic cells, Ca™ binds toa calcium-binding protein called calmodulin, snd the complex binds tothe Ca pump. Other Ct binding proteins include troponin C and parvalbumin.Fig. ar 4 Membranes III ORE ei cu ae Nice ee es rene (04g. Bradenergic receptor), ‘R= regulatory ubunt C= catalytic subunit ea CHEMICAL COMMUNICATION Endocrine signalling occurs when cells or organs release chemicals corhor? which rave i the bloodstream to target cells or organs, Which recognize them through specific receptors. Receptors are proteins that may be situated on the plasma membrane or inside the Receptor (R) activation of adenyjate cyclase eo outsise we s 2 ~ cell, The receptor has sites that recognize and bind the hormone. and eaction produces a change in the receptor which allows it to pass on tothe cell the message thatthe hormone has hound to it Examples of hormones are adrenaline, insulin, the sex hormones and thyroid hormone, Paracrine signalling occurs when the secreting and target cells are adjacent or close to each other. Autocrine signallinginvolves the release by a cell ofa chemical that acts on the cell which released it. Growth factors are often paracrine or autocrine hormones. Somictimes, a chemical such as adrenaline, is both endoerine and paracrine ‘Chemical signals or hormones acting on plasma membrane receptors are generally H,O soluble (e.g. insulin, adrenaline) and produce relatively fast responses (seconds or minutes), Chemicals acting inside the cell are generally lipid soluble (eg. cortisol, vitamin D), enabling them to pass easily through the plasma membrane. Their effects are slower in onset (hours oF days) because their receptors alter gene ‘expression and subsequent protein symthesis. ‘Receptors detect chemicals. They exhibit the properties of binding selectivity, high affinity, reversibility and effector specificity forthe hormone that binds to them. An example of effector specificity isthe action of adrenaline, which in liver cells causes glycogen breakdown and glucose release, while in neurones adrenals electrical impulse “Membrane receptors are integral proteins that span the membrane (On the extracellular surface is generally the N-terminal domain; inside the membrane isa helical hydrophobic ("H,O-hating”) domain; and the ‘C-terminal domain extends into the eytosol, Membrane receptors may be linked to different sign transduetion systems: (i) G proteins: i) jon channels and (ii) enzymes. ‘A G-protein-linked receptor, adrenaline, binds o several different receptor subtypes, termed 4, @,, B, and B.. For example, the B.-adrenergic receptor recognizes both adrenaline and the neuro- ‘ransmiter noradrenaline, as well as several artificial compounds, such as isoprenaline. The B.-adrenergic receptor message transduction system has been relatively well characterized. The receptor itself has seven membrane-spanning holiees, whose arrangement within the ‘membrane may dictate the specificity with which the receptor binds the chemical After the chemical has hound, the receptor interacts with other Separate membrane components, which are the G proteins and the enzyme adenylate cyclase ‘may generate an SECOND MESSENGER SYSTEM ‘Tightly hound tothe eytoplasmic surface ofthe membrane isthe integral G protein, so-called because it binds guanosine triphosphate (GTP) “with high affinity. The G protein consists of three subunits, called a. 8 ‘and , of molecular weight about 42, 35 and 10 KDa, respectively. The ‘subunit can bind guanosine diphosphate (GDP) and GTP. The third protein integral othe membrane is the enzyme adenylate eyclse, which has an ATP-binding site on the cytoplasmic face ofthe membrane an, ‘when the enzyme is activated, it converts ATP to cyclic adenosine monophosphate (cAMP). Tn the absence of hormone, the G protein binds GDP, and adenylate cyclase is inactive. But, when the hormone binds 1 site on the receptor, the receptcr’s conformation is changed and it binds the G protein, GDP dissociates, allowing GTP to bind instead. Consequently. the G protein dissociates into Gy.- and G,,subunits. The G,-subunit binds to adenylate cyclase, which is activated, and converts ATP (o ‘cAMP. cAMP isa so-called second messenger, relaying tothe cell the fact that the hormone has bound to the receptor. Adenlate eyclise activation is rapidly terminated by hydrolysis of bound GTP 1 GDP, resulting in the resetting ofthe system for further stimulation, ‘Within the cell, cAMP initiates a cascade of protein phosphorylations by binding to a protein kinase, When cAMP binds to it, the kinase dissociates into two subunits, one regulatory and the other catalytic. ‘The Kinase is then able to phosphorylate other proteins by transferring the terminal phosphate group of ATP to serine, threonine or tyrosine residues ofthe substrate protein. The end result ofthe cascade may be. for example, glycogen breakdown in liver or muscle, the hydrolysis of twiacylglycerol to fatty acids and glycerol in adipose (fat) cells or the synthesis of steroid hormones in the adrenal core. ‘This type of eascade is an extremely efficient system for amplifying the signal, since the binding ofa single adrenaline molecule results in the activation of adenylate cyclase molecules, and the generation of many molecules of cAMP, Inhibitory G proteins also exist in the membrane, and these are activated to inhibit production of CAMP. They are activated by different receptors and hormones. These G proteins have G, subunits, but 2 different G, subunit, called G,, which binds GTP when activated, but which somehow inhibits adenylate cyclase. For example, adrenaline, through its Bereceptor, stimulates cAMP production, while the neurotransmitter adenosine, through one of its receptors, the A, receptor inhibits CAMP production. The adenosine A-receptor, however, stimulates cAMP production. ‘Nove: cholera toxin, produced by the bacterium Vibrio cholerae irreversibly activates G,. so that it cannot hydrolyse GTP to GDP, resulting in continuously raised cAMP in the intestinal epithelium cell ausing a flood of Na" and H,0 into the intestinal lumen, massive iarhoea and possible death from dehydration, Other examples of second messengers are inositol trisphosphate UP,) and diacylglycerol (DAG), which are generated by activation ‘of membrane receptors by hormones, for example through the action fof adrenaline on receptors, or the neurotransmitter acetylcholine ‘on muscarinic cholinergic receptors. The activated receptor binds to 4 membrane G protein, which stimulates the membrane-bou! ‘enzyme phospholipase C (PLC) to hydrolyse phosphatidylinositol 45-bisphosphate (PIP. w DAG and IP, IP, diffuses into the eytosl and binds to a receptor on the ER, which discharges free Ca into the ‘eytoplasm, The fons facilitate intracellular processes such as vesicle ‘exocytosis oF glycolysis5 Intracellular receptors and receptor antagonists Intracellular receptors Membrane receptor antagonists | poe an ee aries area tet be Intracellular rag r # Receptor Cel receptors soon | 1 ) The Zee 5 / sw ; QUO Ame a i Neen | || to a se eect ee sar (protala DNALbinding ‘Membrane: | santa oe pont rae went teeta agonist Members of the steroid a ‘Signal receptor superfamily i af Ghicocontioaid a nay 4 62 65 TE nescence Receptor antagonists a 8s HH ‘pedrogen a He ae nae as Se raincas eh EE Berroid hormone ‘Numbers abou biocks gi amino elmeeeeee ne ‘the glucocorticoid receptor (4) Pe INTRACELLULAR RECEPTORS. Mechanism of receptor activation Intracellular receptor proteins bind tothe lipophilic hormones which The mechanism of intracellular receptor activation by hormone is not pass easily through the cell membrane. The intracelul receptors known, but there is evidence that i the in hhormone form part of large bound by a heat shock prot so-called receptor superfamily, whose members bind to the nuclear chromatin and alter tanseription, tive sa (HSP90). The number size of the protein. The term ‘heat shock’ was gi proteins were originally he receptor is for steroids, vitamin D and thyFa 10 refers tothe n because these ected after cells were injured by heat shock,although it is now known that they are expressed in untraumatized lissues. There is a family of HSPs associated with diseases, fever, ischaemia, ageing and the inflammatory process. Some HSPs are also molecular chaperones (seep. 107). ‘When the hormone binds to the receptor, the HSP9O dissociates from the receptor, and the receptor proteins form homodimers. The receptor hormone complex binds to specific sites on the DNA, called hormone response elements (HIREs), upstream from inidation sites. Many ofthe deoxynucleotide sequences of the HREs are known, Receptors forthe sex hormones, and for ghicacortivoids such as cortisol, are associated with HSPs, although receptors for thyroid hormone and vitamin D are ‘not, an these receptors appear able to bind to their HREsin the absence fof the bormone. In all eases, the process of receptor activation involves Phosphorylation ofthe receptor, although the exact mechanism su [Nature of the receptor "The members ofthe intracellular superfamily of roceptors contain three main regions. The frst is a DNA-binding domain (region 1) which consists of two “zine fingers’, so-called because each binds an ion of zine (Zn). This region istic in cysteine and basie amino acids. Its ‘lieve that the First zine Finger determines the specificity ofthe binding ‘of the receptor to DNA. while the other stabilizes the receptor 1 is response clement on the DNA. Regions 2 and 3 ofthe receptor determine the hormone speciticty ofthe binding rection. From the scheme shown in Fig. 5.1, itcan be seen that region 1 is very highly conserved among members ofthe superfamily, while the hormone-binding regions show ‘much less homology. RECEPTOR ANTAGONISM ‘The specificity of the binding reaction between ligand and receptor «eats many opportunities For designing drugs that will essen o prevent the action of the ligand. Receptoe-blocking drugs play large part in therapy, and examples inclode: (i) the Bereceptor blocking drugs, for example, propranolol, used (o teat cardiovascular disorders: and (ii) the anticancer drug tamoxifen, which inhibits the binding of the sex hormone oestradiol tots intracellular receptor, ands used in certain {form of breast cancer. ‘Antagonisis can block hormone effects by binding directly to the receptor, o through indirect means. In classical pharmacological terminology the ligand that activates the receptor is termed an agonist, ‘nd the lgand that blocks the action of the agonists termed an antagonist Membrane receptor antagonism In the sehieme shown in Fig. 5.1, agonist 1 binds to its site in onder 10 clicitthe response, while antagonist | binds 10 an allosteric site onthe receptor to block the action of agonist 1. An example of such an ‘agonist-antagonist pairs the neurotransmitter glutamate, wich binds to its site inorder to open fon channels, and the antagonist 2-amino- phosphonovalerate, which binds to an allosteric site on the same receptor to which glutamate binds ‘Two molecules of agonist 2 are required to bind in onder to elicit a response, and antagonist 2 blocks by occupying the sites. An example ‘of such an agonist is aeetyleholine, which binds to wo sites on the nicotinic ceceptor on skeletal muscle fibres. The antagonis ‘tubocurarine, a muscle relaxant, occupies both sites and blocks the action of acetylcholine. ‘Antagonist 3 does not directly antagonize the action of a ligand, but is able to penetrate the membrane and interact with the pos Feceplor mechuaisms wo block the action of the agonist. may, for ‘example, inhibit the passage of fons through @ channel opened by the agonist. An example of antagonist 3 is the anticonvulsant drug phenytoin, which blocks the transmission of ions through the membrane Intracellular receptor antagonism Intracellular hormone action can be antagonized by substances that interfere with the normal hormone-feceptor interaction and pos-receptor binding processes. The receptor itself may be blocked, or the post- receptor-mediated events, for example DNA binding, transcription or translation, Dimerization block Drugs have been developed that block the dimerization of the receptor ‘once it has been activated by the hormone. An example of such a drug is the compound ICLI64384, which appears to block the dimerization ‘of the vestrogen receptor homodimers ater they have been hound by ‘estradiol. (ICI is the company that developed the drug.) Failure to dimerize will compromise the ability ofthe receptor complex to bind to the BRE, ‘Transcriptional block ‘Transcriptional block is the mechanism whereby two importa antagonists of steroid hormone action exer their effects. Tam- oxife and the controversial substance RU486 [developed by the company Roussel to block the actions of the hormone progesterone, and thus terminate pregnaney}, both inhibit activation of transcription activation sites after they are bounel by the receptor ‘mentioned earl6 Molecules | Deu Lory CHEMICAL BASIS OF CELLS be ‘lassifed in terms of funtion and structure. Functionally, cells rl All cells depend on chemical activity, which ean comvenien ‘on chemicals to provide energy, to maintain electrochemical gradients across membranes and 10 provide the messengers which conve information, Structurally, cells tequite chemicals to provide the building blocks forthe structures which have been described in previous chapters, and for growth and repair of these structures. ‘Chemical size (Chemicals can be classified broadly in terms of size, a small oF Lary molecules. Small molecules consist, generally of less than 50 atoms, with molecular weights mainly less than 1000, and have been refered to as metabolic in as Ne srmediates, They include the inorganic ions such stugars such as glucose, amino acids, for example glycine, nous bases including the purines and pyrimidines, fatty ai and steroids. Larger molecules are called macromolecules. In the cell, macromolecules ate the polysaccharides, proteins and the nucleic acids Macromolecules are composed of several smaller molecules, ed chemically by covalent bonds, Wh of one type the smaller molecules are ‘a polymer, and the smaller jonomers, Thus, proteins are polymers. the macromolecule is calle ‘molecules are termes composed of monomers called amino acids. Monomers are also ‘commonly called residues. Nucleie acid ate polymers of nucleotides, es are polymers of sugars. Some proteins contain and polyssce! polysaccharide moieties or groups covalently attached, when they are called glyeoprotein, NUCLEIC ACIDS Nucleic acids are composed of nucleotides arranged in immense chain, Each nucleotide in tum is made of a base. either a purine or 8 pyrimidine, a pentose sugar (Five-carbon sugar) and a phosphate group, "The sugar is ribose or deoxyribose. When the sugar is ribose the 1 is deoxyribose the nucleotide is DNA, andthe nucleotides are deoxynueleotides. DNA, together with essociated basic proteins called histones, makes up the chromosome Humans have 46 chromosomes arranged in 23 pairs in the cell nucleus, and these contain the hereditary information of the body. The cluromosomes have streiches of DNA, called genes, which code for specific proteins, as well as having associated sequences of deoxy nucleotides wihich contol the expression ofthe genes with which they are associated, A cell that contains two sets of the chromosomes is, termed diploid, while a cell that contains only one set is termed hhaploid. Humans are therefore diploid organisms. The human cell, whether spermatozoon or unfertilized ovum, has only one set of 23 chromosomes, und is termed haploid, Whon Fertilization, the diploid status ofthe cell is restored wo combine duringCP ‘Adenosine triphosphate (ATP) Me a nucleot! BK Sun roy Se ana Nucleotide structure For DNA, the bases of the nucleotides are ring structures, ether pyrimidines (thymine (T) or eytosine (C)) or purines (guanine (G) for adenine (A)). For RNA, the thymine is replaced by uracil (U). For both DNA and RNA, the nucleotides an rough the sugars, where phosphate groups li ‘one sugar to the 5” point of another, releasing & molecule of HO. med 3'5’-phosphodiester bonds, In this way, a linear chain of nucleotides is built up. Because there are four different nucleotides, and because the chains can be assembled in any order of nucleotides, there are 4° different possible nucleic acids having nucleotides. For example, a nucleic acid liked (or ‘eondensed) the 3° point of These phosphate linkages are t. containing 15 nucleotides has 4" possible combinations (more than 1 x10), The chain is represented as a strand with the 3” end having. fn unsubstituted hydroxyl (-OH) group on the left, and the phosphorylated 5’ end on the right, During synthesis of DNA (or RNA), nucleotides are added to the 3” end. ‘a | Qy ala] So) ‘Shorthand for DNA Base pairing: thymine and adenine aa 1 aes HA Ana. Cis roc he a | ee seo swing Le Hi HHO Fig 2 Base pairing and the double helix The DNA mole: intertwined nucleotide strands. The strands are held together by what is called base pairing, When two strands of DNA come together, guanine will pair with eytosine, and adenine sill pair with thymine throu son-covalent hydrog s in the cell consist of wo complementary en bonds. Thus, the base sequence of one strand ‘will always be complementary to tha of the other Also, due tothe Way DNA is synthesized, the paired chains run in opposite directions the 3° adjacent to the 5” end ofthe other, Structurally, ‘the chain forms what is termed a double helix. The double helix is form of twisted or spiral righthanded staircase coiled round an imaginary central core. with about 10 bases every turn and the helix makes a complete turn every 3.4 nm, The double helix is held together not only by hydrogen bonding but also because the bases are planar and form stacks of base pairs held together by hydrophobic and van der Waal’s forces (see Glossary, p. 112). The pairs can be made to separate end of one chai I simply by heating the DNA, when it ‘melts’ or denatures.7 Molecules II OEE ued A ‘ voce {e— 1m wooo-Zt— ni fs 4 PHO yeas, feet ee h 4 ‘Condensain of wo amin acide and eatin of apenige bord PROTEINS Prot perform many functional ra also depends absolutely on proteins for its structure. Unlike DNA. ny three-dimensional shapes, depending on theit ins are polymers made up of monomers —amino acids. Proteins inside and outside the cell, which utning acid composition and arrangement. They may be fibrous (.2 fibrin, collagen), globular (e.g. serum hormone-binding proteins), or antibodies, Amino acids are so-called because all, with one exception, contain amino (-NEL) groups, The exception i proline, which contains an imino (CNH-) group. They also contain acidic carboxyl (COOH) groups. (Chemically, amino acids ha atom lying adjacent tothe acide carboxyl group is attached to (i) the (or imino) group; (i) a hydrogen atom: and (iv) toa variable ed, the charge common design (i) a central (-earbon side chai (R). At neutral pH, amino acids will be depending onthe relative numbers of amino and carboxyl groups. Lysine ispositvely charged, while aspartate is negatively charged. Some a ocids are more hydrophobbe than others, depending on the hydrocarbon content of the R side chain, All amino acids, except glycine, have asymmetrical carbon atoms, and can therefore exist as stereoisomers, ‘The two forms are miror images, termed p and, and proteins virtually always contain the isomer. mino acids condense into polymers through the peptide bond, whieh joins the amino g imino acid tothe carboxyl group of another, with the release of a molecule of H,0. If the amin med & peptide, polypeptide. Polypeptides can range in amino acid number from theee is less than 30 residues long. it is commonly (e.g. glutathione) to those whieh are made up of more than 1000 amino acids. Every polypeptide has a free amino group at one end, aod a acboxyl groupat the other. Proteins contain 20 different ypes of amino acids; therefore, « 24)-amino acid polymer can have 20° possiblestructures, This offers enormous variability in protein structure and function. Proteins have four structural levels: 1 primary — the linear sequence of amino acids and the S-S bonds: 2 secondary — protein folding into a-helix and pleated sheets: 3 tertiary — regional folding between pleated sheets and e-helix, ‘determined hy non-covalent bonds; and 4 quatemary — non-covalent binding of different polypeptide subunit chains into a single protein molecule (e.g. haemoglobin, Hb), Polysaccharides ate polymers of sugars (also termed saccharides). Examples of polysaccharides are starch (storage form of glucose in plant cells), cellulose (par of plant cell wall) and glycogen (storage form of slucose in animal cells). Linkages ean occur several different ways, and polysaccharides can occur in many different branched forms.8 DNA replication Ose Replication in E.coli DNA chain elongation at 3' end soe z ease 5 | ONA payraase | : DuAToee ea NA pine ‘S888 = Snglestane binding prtein irectional replication of circular DNA ote * Lox7 nc o ° } we arcs, i in| 0 aaa Bon H sruceotde | Fig at DNA replication: (i) DNA chain synthesis; (i) its initiation; Gi) and replicated bidirectionally, until eventually all replicated stretches termination; (iv) packaging with chromosomal proteins: (¥) recom bination: and (i) DNA repair Anew strand of DNA combines with an oid one, This i semmi-conservative replication dirough growing forks, Replication s bidirectional, with both strands being copied. Each copied region is termed a replicon. Eventually, the replicons merge. In cukaryotes the double helix is unwound simultancously at several sites, join up to form the n is initiated by an appropriate cellular si 3» complementary DNA (cDNA) strand. al. A helicase enzyme system hinds toa specific nucleotide sequence and unwinds the DNA adjacent to ittocreate a replication fork, The DNA is unwound just ahead of the DNA polymerase moving up behind to replicate the avvindin ‘exposed strandQOQ0G002 — e343 e> | a i) u REPLICATION Replication is initiated by synthesis ofa primer sequence of RNA at the starting point of replication on both unwound strands by primase enzyme activity of DNA polymerase (The primase gene is termed. dua.) The polymerase begins elongation a the ¥ end ofthe primer. Elongation proceeds continaously along the S'—> 3’ leading strand, ‘On the so-called lagging strand, clongation must also rn fom $°—> 3", and runs inthe opposite direction. Therefore replication onthe lang strand is discontinuous, and has to stop periodically to wait for more “ofthe strat unin. This creates segments of cDNA, called Okazaki Fragments about 100-200 deoxyribonucleatdes long in eukaryotes. “They are formed when proteins called primosomes attach themselves to the RNA-DNA complex and activate the reaction ready for the oJ ‘ee polymerase After formation ofthe Okazaki fragment the primers are temoved and replaced by DNA, and the fraginents are joined together ‘by a DNA ligase. Errors in the matching of bases during elongation are detected by DNA polymerase 8, which ‘proofreads' the bases. In eukaryotes, DNA is associated with nucleoprotein histones. Their basic charge enables the histones to hind tothe phosphate backbone of DNA. The nucleosome isa disc-shaped coil of DNA wound 1.5 times ound a cluster of proteins including two molecules each of H4, H3, HB and H2A. The polynucleosome consists of several nucleasomes joined by linker DNA. "The chromatin formed from the DNA-protein ‘complex is condensed into chromosomes. Fig a9 DNA repair ‘Action of DNA ligase| | A | t | 1 ‘comm 5 | ir I “he mh DNA REPAIR DNA repair is the maintenance of DNA stability through removal of cerrrs in base sequence due to chemical or rradiation-induced damage or through errors introduced during replication. Stability of DNA is species would he jeopardized by errors in rapidly dividing germ cells, DMA ropa it 1. Urae DNA goosyase 2. AP endonuclease ‘3. DNA poymerase + ONA nase tag O pra 5 y sie co. co. AT AT Ethidium bromide ay TA. ca. [Tiwinor oa] ‘rama si, hen TASH toni Bardi ages ring adaton | Base ising pues removed by acd of eat Base corec-C > U:A—> nypoaning ] Break stan ony R | inserien don rain ages Cyst dir: UV eran Sard cose tnkng -mitomyenC ° Me Ne i i 1 ae = ore Ne i a _ _ posing satan cause: Chemica raion: recicaton eros and cellular function would not be possible in stable cells, such as brain ‘or liver cells, which may not divide for years In Escherichia coli, DNA polymerase 1 introduces on average one incorrect base per 10° hase pits. Genes in E.coli are about 10° bases long. Therefore, ertors would be introduced every tenth gene (10" errs (or mutations) per gene each generation). But, the actual rate of errors is much less in E coli: around 10 errors per gene each generation. Its‘thought tha the rate of eror production is similar in animal cells, whieh must also have a mechanism for repairing errs in coding regions of the genes. ‘Mutations canbe defined as stable alterations in gene DNA structure, “They may be silent, or expressed as phenotypic alierations, Mutations ‘may be one ofthe following. 1 Brame shift mutations, which result fom: (4) deletion of a base pair or block of base pairs: (b) insertion of new base pairs 2 Base substitutions, which esult from: (a) transversions, which are the substitution of pyrimidine-purine ‘ase pairs by purine-pyrimidine base pairs (b) transitions which are the substitution of purine-pyrimidine base Pairs by pyrimidine-purine base pars. ‘Tests for potential carcinogens include tests for their mutagenic effects on bacteria. This implies that alterations to DNA lie atthe ro0t ‘of both carcinogenesis und mutagenesis. One testis the Ames test, in which a strain of Salmonella, unable to synthesize histidine, is grown, ina Petr dish ina medion that lacks histidine. Any bacteria that grow are natural mutants. The suspected mutagen isadded, and many mutants may be formed — some of which can synthesize histidine — and they ‘will appear as yet more visible colonies on the agar. Different strains have been identified, some of which respond to mutagens that cause hase substitutions, while other stains respond to mutagens producing ‘adtionso¢ deletions. frame shifts. This makes it possible to identify, tentatively, the mechanism of action of the mutagen/carcinogen, Repair is elTected by proofreading. The proofreading ability of DNA polymerases was first discovered in E.coli, The enzyme DNA polymerase 1 is believed to have 3” ~> 5’ exonuclease activity, which enables it to proofread newly added base sequences, excise non- matching bases and replace these withthe eoreectly matching bases. Repair of pyrimidine dimers Pyrimidine dimers are formed after exposure of DNA to ultraviolet (UV) light, when adjacent pyrimidine residues on a DNA sttund may become linked covalently. The distortion of DNA produced by the dimer is Uetected by a group of prowins expressed by the wvrABC genes: the proteins consist of a tetramer of 1Wo proteins, P,.. and swo proteins, Py The exonuclease enzyme P< cus the strands at two places: four rcleotdes away from the dime* on the 3” side, and eight nucleotides way on the 5’ side. The excised 12-residue piece of DNA is unwound by a helicase P,, and diffuses away. DNA polymerase | maves into the gap created, and uses the 3 cut end as the primer and the intact complementary strand as the template to repair the cut strand. Finally. DNA ligase joins the 3° end of the newly synthesized DNA and the original DNA. Excision repairs also used by the cell to remove ctosslinks formed bby drugs used to teat cancer, such as explain, mitomycin C and the nitrogen mustard ‘Note: E coll contains an enzyme, DNA photolyase, which binds to the DNA region distorted by the dimer, and becomes photoactivated and splits the dime. Repair of deaminated cytosine ‘Cytosine may be deaminated to uracil, nd this occurs spontaneously ‘during the life of DN. Since uracil can pair with adenine (UA), the ‘chemical change is potentially mutagenic. The presence of uracil on the DNA is recognized by uracil DNA glycosidase, which breaks the bond between uracil and deoxyribose by hydrolysis. The gap formed by removal ofthe pyrimidine is called an AP site (i.e apurinic, containing no cytosine or thymine. The gap is recognized by the enzyme AP ‘endonuclease, which euts the DNA backbone adjacent to the missing base, DNA polymerase Leuts away the piece of deoxyribose phosphate and inserts cytosine opposite the intact guanine residue on the ‘complementary strand, and DNA ligase seals the cut DNA strand, Diseases associated with defects of DNA repair These include hereditary retinoblastoma, Fanconi derma pigmentosum. eroderma pigmentosum isthe best understood. This rare disease fs genetically transmitted as an autosomal recessive tat, Patients are highly sensitive to UV and sunlight, developing skin lesions soon after birth. The dermis atrophies, the eyelids sear andthe cornea uleerates, Freckles and skin ulcerations appear, followed by skin eancers. ‘The disease is caused by a defect ofthe exonuclease enzyme which nicks the DNA atthe site of pyrimidine dimers, which are known to be ‘caused by UV radiation. Skin fibroblasts from patients with Xeroderma pigmentosum have been shown to contain the deficient enzyme, Mutations in one of atleast nine different genes can cause the disease Although the incidence of the disease is low, about 1% ofthe population are cariers of a least one of the mutated gene. naemia and Xero10 Recombination Co Soo, = =e 2ee Heteroduplexes oe Recombination and heteroduplexes | DNA. This‘Note: alternative Forms of a gene, termed alleles, can be exchanged ‘by general recombination al ean occur on the same chromosomal MECHANISM OF RECOMBINATION Initially, strands of DNA can join to each other by non-covalent base pairing to form lap joints, and these joins are made permanent by subsequent DNA synthesis. ENZYMES OF RECOMBINATION Single-stranded DNA for recombination is generated by a complex of proteins, P_, P.,eand Py, Which ae products ofthe recB, recC and ‘reeD genes, and which form a complex provein enzyme system. In -eoli the complex: (i) recognizes sequence — §-GCTGGTGG-3' — the so-called chi sequence, and cuts the strand about four 10 six nucleotides away from the 3 end of the chi sequence; and (ii) the complex unwinds the DNA strands, The process requires ATP hydrolysis, In E.coli the recA protein utilizes ATP o catalyse the assimilation of a single stand of DNA into a duplex, The protein: (i) binds to the Single strand, and the protein-DNA filament thus formed binds 1 the i) partially unwinds and ‘reads’ the duplex for sequences ‘complementary 10 those on the single strand: (i) further unwinds the duplex where complementarity and pairing oceurs; and (iv) the exehange process continues along the duplex, and these are the mechanics ‘underlying the process of branch migration mentioned above, All these ‘steps require the hydrolysis of ATP. SOS RESPONSE Normally, low levels of P.., are present in E.coli, due to suppression ‘of P,., MRNA production by a repressor protein termed Pray Pay suppresses the production of many repair proteins. When DNA'Is ‘damaged in E: coi, the cell initiates what is ealled an SOS response, generating hundreds of new copies of recA protein: (i) damaged single stranded DNA binds to existing recA protein; and (ithe complex formed binds to lexA protein and splits its alanine-glycine bonds, rendering it inactive, Note that P_, therefore acts not only asa recombinase but alsoas protease because of ts action on P_,. Another gene activated by Inactivation offexA isthe wv gene, whose product the usrA nuclease, cuts away thymine dimers which are formed by UV radiation damage. MOBILE G NETIC ELEMENTS (MGEs) GES, abo called transposable elements or transposons, mediate the large-scale rearrangements that cannot be effected by general recombination. Plasmids are important MGEs confined to pro- karyotes, Plasmids are circular duplex DNA molecules which can replicate autonomously, and are therefore replicons. A replicon is any ‘unit ength of DNA which possesses its own origin of replication, and includes plasmids, bacterial chromosomes oF regions of eukaryotic DNA Plasmids carry genes which express: () the generation of bacterial toxins; (i) the metabolism of metabolites and other chemicals; and Gi) the inactivation of antibiotis. They are therefore tols of evolution tnd adaptation, Plasmids ate also tools used by genetic engineers to introduce novel genes into cells. Engineers have termed particles such as plasmids, which are used to wansfer genetic information between cells, vectors, In recombination, plasmids may be plasmids, factor plasmids or factor F factor plasmid ‘The DNA sequences of F* factor plasmids (1 bye transferred between bacteria as follows, 1A ‘male’ bacterium (F*) fastens itself oa “female” bacterium (F) by means of a ex pilus on its surface, 2 The pilus retracts to pull the two bacteria into close contact; one strand of the plasmid is cut and the DNA duplex wawinds 13. The S’end of the eut strand passes into the “Yemale cell, where 3 ‘complementary strand is synthesized and a circular duplex plasmid formed. ‘The recipient coll is now F* and the transferred plasmid has the genetic information required for production ofa sex pilus ‘The new plasmid can: (3) remain separate from the main bacterial chromosome: or (i) become integrated into the chromosome, when the bacterium is now denoted as being a high frequency of recombi- nation (hf) cell. An hit cell donates not just the F* factor plasmid during transfer, ut its complete chromosomal database. An hfe cell ccan splice out the F factor from its chromosomic, and thus reverts to the F state, factor plasmids are not the only means of transfering genetic information betcen bacteria. Ths information can also be transferred bby MGE called bacteriophages. A bacteriophage isa virus that infests bacteria, stands for Fertility) can R factor plasmids ‘These are so-called because they catry resistance to antibiotics. A bacterium can develop resistance 10 one or more antibiotis if it receives an R factor plasmid. The plasmid may contain a resistance transfer factor (RTF) as well as many so-called r genes. r Genes ‘express enzymes that inactivate antibiotics such as tetraeyeline, streptomycin, chloramphenicol and sulpharila lacking an RTF region, will confer resistance to a single antibiot ‘when transferred ile. Smaller plasmids,11 RNA Base Ribose phosphate | t it Bh \e oT Sas oF ssaace —Tanlandsogone PAIR | ion eet ‘S 0 CHa mi structure | i | oO Oya we ov yn . nto ste id i oF ON / 0 CHa ~~ 4 : oO amis Ny x wyhoety Nn x 3’. start sites, RNA polymerase II requires three start sites at ~110 istheCAAT box (CA-CAATC); at 40 isthe GE box (GGGCGG): at-25 is the TATA box, also called the Hogness box (TATAAA, The CAAT box aids the binding of polymerase Il to the DNA. and the TATA box guides polymerase II to the core sat site. The GC bor is most often found on constitutive genes (continuously expressed), rather than on those which are, for example, developmentally regulated. In addition to promoters, there are enchancer sequences which ‘may be thousands of bases away from the start site, either upstream, {downstream or both, and may be on either coding or template strands of the DNA, Enhancers on their own, however, are not promoters Enhancers xy confer specificity of cellular or organ responses to chemical stimuli. They have sites that bind sets of protein modifiers, ‘both positive or negative in action. Steroid and thyroid hormones, for example, bind to intracellular receptors which in turn bind 10 enhancers, thus initiating transcription, Only those cells that normally ‘contain the receptors and/or the enhancers will respond to the chemical stimulus, TRANSCRIPTION FACTORS ‘Transcription factors are proteins that are required in onder for RNA, polymerase to recognize promoter sites, or example, genes containing GC boxes require a protein termed spl A transcription factor called CTF binds to the CAAT box, and a protein, B protein, discovered in Drosophila binds to the TATA box. ‘The 5" cap is added to the 5” end of eukaryotic mRNA very soon after transcription is initiated. The cap has at leas three functions 1 it protects mRNA from enzyme attack: 2 itis important in subsequent splicing: 3 itenhances translation of the mRNA. ‘The 5" cap contain an “inverted” base, 7-methylguanylate, attached ‘o methylated ribose unit The 3 polyA tall of mRNA has an unknown function. It is not ‘encoded by the gene and is added afte cutting ofthe primary mRNA, transcript atthe cleavage signal AAUAAA, which is recognized by a specific endonuclease enzyme. The polyA tal is not required for transcription, and some species of mRNA do not have a polyA tai SPLICING Splicing isthe removal of pat, if not most, ofthe newly symhesized mRNA precursors. Only the mRNA corresponding to DNA exons are needed for transcription. Exons are the regions within the eukaryotic ‘genes that are expressed, and inthe DNA are separated from each other by itrons, which are non-coding regions of the DNA. mRNA corresponding to the DNA introns is eu away from the pre-mRNA by specie splicing enzymes, All eukaryotic systems described so far have introns that begin with SGU and end with AG-3. Eukaryotic DNA, contains many non-coding regions of repetitious DNA, which has been called ‘junk DNA’ The site of splicing is detormined by the 5" and ¥ splice sites and 2 region ofthe intron called the branch site. The branch site i a short, sequence of ribonucleotides which may vary with the cell type and the species. All thre splice sits, ic. 53” and the branch ste, must be in order for correct splicing, Mutations ofthe sites can cause disease due to incorrect spicing. ‘The mechanism of splicing requires (i) the culting of the 5 splice site: (i the cutting ofthe splice sit: Gi) the release of the intron in lariat’ Form (so-called because it resembles the cowboys" lasso); and (iv) the joining ofthe wo exons which are on either sie of the spliced intron, The two exons ae joined by means of two transesterification reaction, in which the free -GH group of one exon is Finked to the free 5’ phosphate of the other. While these steps are heing carried out, the whole assembly is held together by the splicosome, ‘The splicosome is a relatively large 60S complex made up of the mRNA precursor and three different species of small nuclear RNA (6oRNA). Molecules of snRNA have heen nicknamed ‘snueps’ (smal cytoplasmic RNA has been nicknamed ‘scurps’), Different snueps have iferent functions in spicing Snurp onkNA) | Function in splicosome v, Binds ¥ splice ste Binds branch site Binds ¥ splice site Assembles the splicosome Catalytic RNA (self-splicing) was discovered in protoroa, in which RNA can splice itself in the absence of any protein, ie. enzymes. ‘Catalytic RNA is stable, and appears to function like an enzyme. Ir has been shown 19 catalyse the cleavage and joining together of ‘other nucleotides: Its therefore both a ribonuclease and an RNA poly merase. Furthermore it exhibits the features of enzymes, being highly ‘elective for substrates, obeying saturation kinetics and the kinetics of ‘competitive inhibition, Sel-splicing RNA is important evolutionarily, since biochemical reactions of DNA and RNA couldhave taken place even before the evolution of protein enzymes involved in DNA and RNA synthesis14 Errors of transcription SER Hormone resistance | Trascigfona Ftanpicn sons cause requis and ‘nes RNA ‘rare sits 9 erectary olmerase of ‘goin coding retrepastera Mores sequence SOCTATTOGTC sneseraTTAGGG eevee 3 normal fbi non we SCCTATTAGTC sneeenaTTAGGS coves 3 fli intonn lassaomia (G+ mutation cates raw spice sie NA — breing = Homonetinsng | oman onan ‘sated reer eave ting of emane agrarmate reap siete | captor coords a ‘taninD ‘Toyo termane INTRODUCTION CANCER Errors of transcription can result in disease, and transcription is vulnerable 1otoxie agents such as e-amanitin, and wo certain antibiotics. Inappropriate expression of transcription and growth Factors ‘cancer, Mutations of the DNA can cause changes in the location of splice sites, wth resultant defective functional and structural proteins. ‘These mutations can result in defective receptor proteins fob The vulnerability of transcription to drugs such as fungal toxins and to antibiotics means that these are useful both as research tools and as potential chemotherapeutic agents The comersion of normal cells into malignant cells is mediated by. for pe, viruses, matagenic chemicals an ionizing radiations. Malignant roland may kil the ost organism if not stopped growth factors and reduce cells divide out of “Malignant cells ma ‘or abolish their susceptibility ton ‘Transforming growth factors (TGFs) are chemicals that can convert prodicin ative growth factors, that toa malignant cell. Por example, there is evidens the hormone pro called TGF-c, which may mediate the growth of breast cancer cells. In one increases the expression ofa growth factoranother type of cancer, hereditary retinoblastoma, a rare cancer of the retina in infants, the cancer cells lack the Rb gene, which in normal ‘ells may be a negative regulator of transcription. ANTIBIOTICS ‘The antibiotic rifampicin, a semi-symhetic derivative of rifumycin, whieh was isolated from Streptomyces, blocks the formation ofthe frst ‘phosphodiester bond in RNA synthesis (se also p.25).Cain elongation, however, is not affected. The bacterium that causes tuberculosis is ‘Mycobacterium tuberculosis, anditis highly resistant to most antibiotics, Bt, i is suscepuble to rifampicin, which is not as toxic to mammalian RNA polymerase, Rifampicin is used together with an antimetabolit, isoniazid, to weat the disease THALASSAEMIA “Thalassaemia (Cooley's anaemia) isan hereditary blood disease, especially, prevalent in Aftica, Asia and the Mediterranean counises, in which the slobin part of the Hb molecule is altered, with decreased synthesis of the ce or Brchains. The geographical distribution ofthe disease coincides largely with that of malaria, Patients with the disease are anaemic because the Hb molecule cannot function normally. There are wo types (thalassaemia major, when the disease is inherited from both patients (Ge. there are two copies of the abnormal gene) andthe patient is badly affected: and (i thalassaemia minor, when the defective gene isinherited from one parent. and the child has mild symptoms. or is free of symptoms. In thalassaemia major, affected individuals are anaemic and have swollen spleens and abnormalities ofthe bone mars, They may require blood transfusions which can result in an iron overiod. ‘The disease can also be classified as a-thalassaemia or Bethalassaemia o-Thalasssemia is a deficiency ofthe c-alobin chains ‘due to unequal crossover between adjacent c-alleles, Thalassemia, \which is rarer, ean be due o one of a number of different mutations. For example, there is a G+ A mutation of the B-globin coding region, resulting ina new spicing site: thus, a frameshift occurs. The promoter ‘may be mutated, or the nascent mRNA prematurely released from the template strand othe splicing may be i HORMONE RES NCI Steroid and thyroid hormones, and vitamin D all act on their target cells by combining with intracellular receptors, The hormone-receptor ‘complex binds o specific sequences upstream of transcription start ‘Sites and triggers transcription, The receptor protein has two impertant «domains: one that binds the hormone and another tht binds the DNA. ‘This knowledge, and the knowledge ofthe structure ofthe genes that ‘code for these receptors, has Jed to an understanding of the eauses of Several diseases characterized by a lack of responsiveness to these hormones For example, androgen resistance isthe failure to respond to the male sex hormone testosterone, and to its powerful androgenic ‘metabolite So-dibydrotestosterone (DHT), The disease may be caused by: () the complete or partial deletion of the gene that codes forthe androgen receptor; (i) splicing defects; (if) premature termin: ‘eadons; and (i) amino acid substitutions resulting from mutated base substitutions, These mutations usually mean a loss of affinity of the hormone forthe receptor lathe case of androgen resistance, most of the amino acid substitutions oecurin th steroid-binding domain of the receplor “Thyroid resistance, to0, can be caused by mutations of the gene that cates forthe thyroid receptor. These patients have retarded growth and lesions of hone, despite having high plasma levels of thyroid hormone, ‘The issues are not ‘seeing' the hormone, Two distinet thyroid receptors ‘cand, are encoded by two distinct genes, the a- and f-genes. Incases ‘of generalized resistance 1 thyroid hormone, it has been discovered that most mutations occur within a tightly citcumseribed region ofthe Begene. Glucocorticoid resistance can also be explained, in part, by mut tions of the receptor, and this has important implications for therapy singe glueccorticoids such as prednisolone are used extensively as nt-inflammatory agents in connective ise diseases, and as immuno- ‘uppressans in autoimmune diseases. Thus, patients who ae resistant _lucocorticoids will not espons to treatment with them, In some cases of ‘lucocorticoid resistance, point mutations havebeen discovered resling inthe substitution of single aminoacids This reli ina reduce affinity for the hormone15 Protein synthesis | Cre uy at (4) ein at nino a ‘Arinoacyl RNA sya ATP 6 HO. Me +201 {ANAshottand | Diagrammatic NA Fig. the tramlation ofthe sequence of bases in the DNA, and therefore inthe INTRODUCTION comesponding mRNA. into segue Proteins are synthesized by «process called translation, Tis is literally form he polypeptide chain, the primary structure of proteins. The primary hero mino acid joined to 30structure will determine, in turn, the secondary and tertiary structure, and therefore the Function of the protein. Protein symthesis, asin the ‘ease of DNA andl RNA syuthesis, takes place in tree tage: (i) initiation; (i)elongation: and Gi) termination, Amino acids are activated through Tinkage 1 their RNA molecules in a reaction eatalysed by an enzyme. anaminoacyltRNA synthetase specific forthe particular amino acid In some cases there may be more than one {RNA and enzyme serving ‘particular amino acid, Polypeptide chains are elongated inthe direction “amino to carboxyl group (NH,” -> COO), by the polysome “assembly line’. An initiator RNA triggers synthesis by binding toa ste on the ribosome: elongation begins with the binding of another RNA to another site: and termination occurs when protein release factor ‘reads’ a stop signal on the mRNA. THE GENETIC CODE ‘Atleast 20 different amino acids occur in proteins, but there are ony four bases in DNA and mRNA, and it was discovered that a sequence ‘of three bases called a eodon, codes foreach amino acid. The number ‘of posible triplet codons derived from four bases is = 64. Experiments hhave determined that ofthese, 61 combinations code for amino acids. Although some amino acids, for example tryptophan, have only one codon (UGG), others, for example serine, have as many as six (see below). Nevertheless the genetic code is specific and unambiguous: ‘one codon codes for one amino acid onl. Midate base of codon UC AG Sendo codon Pre Serr Gs Pre Sere Gs Lev Ser STOP STOP A Ler Ser STOP TCG. é Lea Pm His AU ¢ La Po Hs Ag ce Ley Pro Gin AA c Lu Pro Gin AG A Ne Tw Am Sr a Ne Th Am Sere A Ne Te ys Ag OA A Met Th Ls Ag G G Vil Ala Ap Gly G Vil A Ap Gye o Val Au GW Gly A @ Val Al Gy * The codon for methionine (AUG) is & START codon for translation (see ». 30), For definitions of abbreviations sce Abbreviations, p. 108 Because there is more than one codon for amino acids, the code is referred to as degenerate, The code is also virtually universal, since substantially the same code has been found in all living organisms. Codon ‘Mitchondrion Normal meaning cus The Lew AUA Met He uGa To stop Mitochondria provide the only known exception to the rule of ‘universality since some codons which normally have one meaning are different in mitochondria, ‘The START signal for protein biosymbhesis in eukaryotes is AUG, the codon for methionine. Methionine is therefore the first amino acid ‘of the protein chain, The STOP signals, UGA, UAA and UAG, do not ‘code for any amino acids, and may sometimes be refered to as nonsense ‘codons, ‘THE ‘WOBBLE’ HYPOTHESIS ‘The ‘wobble’ hypothesis is an explanation for the fact that: (i) one {RNA can read mote than one codon; and Gi) many of the codons can be read by more than one species of tRNA. Note, however, that fone tRNA can carry only one species of amino acid (i) and (i) may bbe possible because the stringency requirements for base pairing ‘normally stipulated by the Crick-Watson base-pairing rule do not apply, allowing base pairing between the thied position of the codon fon the mRNA reading 5/—> 3’, and the first position of anticodon ‘on the (RNA 5” —> 3, This makes the nucleotides literally wobble. changing the geometry of the codon-anticodon interaction, and allowing G-U base pairs to form. Wobble, and therefore the relaxation of stringency of base pairing between anticodon and codon in protein synthesis, may also be induced by the presence of modified nucleotides at or near the first position of the anticodon in some IRNA species. An important modified nucleotide for wobble is Inosinte acid (1), which can form base pairs with A, C or U in the third position of mRNA codon. The relaxation of stringency of the base-pairing rule during wobble means that, theoretically, many of the different codons could be read by relatively few {RNA anticodons. Innature, however, this possibility has not necessarily been exploited, since most cells contain almost as many species of IRNA as there are of amino acids, THE AMINOACYLATION REACTION Amino acids are activated for protein synthesis, Le. raised to a higher ‘energy level for participation in protein synthesis, through coupling to IRNA in a reaction catalysed by a specific aminoacyl-tRNA synthetase, There arc 20 diferent enzymes; one foreach amino acid. ‘The enzyme ean: i) recognize its specific amino acid-tRNA complex: and (i) proofread the complex after it has bound it, and hydrolyse the incorrectly bound complex. It isnot known with certainty hov the enzyme proofteads the complex.16 Protein synthesis Il UCU lc Al Sytetase Trensfomylase RNA + Met" at —1RA "set —RNA leiatoncompin 708 PYE cto eee (ie (a) 5S - ao ow a ; core «| iy + 1) (1Fa]<— > ¥ SY @— soarcc — 13 — new UU a rm ea a ee ss > oS S. = fe se am L____ jesrna Protein assently 8 cob rhosome = 2 — | omen eeoeb6s Foasore sony pone) in | be tberonaletuntsos] Nell ints] The ribosome re INTRODUCTION THE RIBOSOME nucleoprotein particles. Ribosomes consist of two main subunits which em Proteins, studied. the ribosome is an irregularly shaped ribonucleoprotein particle having a sedimentation cetficient of 708. a diameter of about20-nm and a mass of approximately 2700 kDa. The ribosome can be dissoctated inthe laboratory ina large SOS subunit and a smaller 30S subunit, These subunits can be dissociated further into the proteins and RNA which make them up. The structure and function ofthe ibasome {depends principally on the way the consituent rRNA molecules fold and associate with the proteins. Each bacterial cell has about 20000 ribosomes which constitute roughly 25% ofthe mass ofthe cell. Viewed under the electron microscope, ribosomes may be observed free in the cytoplasm, Eukaryotic ribosomes In mammalian cells, the structure and function ofthe ribosome is very similar to tht ofthe E.coli ribosome, although its composition differs, The marnmalian ribosome is 80S, having a mass of 42kDa, and dissociates into 608 and 40S subunits. he 408 subunit has an 188 4RNA, ‘molecule and about 30 associated proteins, and the 40S subunit is made ‘upof $5,588 and 288 FRNA, and about 48 associated proteins. Viewed ‘under the electron microseope, ribosomes may be observed fre inthe cytoplasm or firmly atached to the BR, Asa general rule, fee ribosomes ‘synthesize proeins for use inthe cytoplasm, while membrane-bound ribosomes synthesize proteins for export From the eell or for making membranes ‘TRANSLATION For protein synthesis, several ribosomes can bind simultaneoulsy to a mRNA molecule to form a polyribosome, or polysame. There may be tp to one ribosome atached every eight nucleotides along the mRNA. ‘The individual ribosomes ofthe polysome work independently ofeach other, and each produces a complete polypeptide chain. Polypeptide sare synthesized in the direction NH, + COOH, an the mRNA {stead fom 510. Ineukaryotes, mRNA is exported from the nucleus tothe cytoplasm, where protein synthesis occurs, whereas in prokaryotes such a6 £ coli, a mRNA molecule may be translated even while its being wansribed. Initiation in prokaryotes Protein symthesis in prokaryotic cells is initiated by the combination, in the cytosol (se p. 30). ofthe free, smaller subunit of the ribosome wit nintiator RNA molecule that caries the amino acid methionine (Med. “The initiator (RNA: (i) is linked with Met through a reaction catalysed by the corresponding aminoacyl-tRNA synthetase: and (it) the Met attached to the RNA is formylated by a transformylase (Toemyl is derived from formic acid, HCOOH). The initiator URNA is therefore expressed as RNA, ‘The Met attached to RNA, does not usually form part ofthe final polypeptide chain, but i removed ater the protein has been made, ‘Note: Met atached to an intator tRNA, can be formylated, but i cannot be formylated if attached to the species of RNA that cart a molecule of Met destined for permanent inelusion inthe protein ln this latter ease, the URNA that carries Metis designated tRNA... iM, ‘The free, smaller 308 subunit binds three initiation factors IF). IF, 1IF2 and IF3, IF2 binds GTP, and also recognizes the Met-tRNA, complex. The reation between IF2 and GTP also enables the mRNA ayolecule to be bound by the 308 subunit. TF3 dissociates 9s the 30S-Met-iRNA, complex is formed: GTP is hydrolysed asthe larger ‘508 subunit joins the complex; and IFI and IF2 dissociate. ‘The ‘Met-IRNA, molecule i located in the Psite ofthe ribosome and the A site is empty atthe start ofthe elongation phase of protein synthesis, ‘The resultant 70S complex is termed the initiation complex. ‘The Shine-Dalgarno sequence “The Met-4RNA, molecule has the unticodon UAC. which binds non covalently to the initiation codon AUG on the mRNA molecule. AUG ceodes for Met. In E.coli the site of initiation is situated at purine- rich region of the mRNA upstream from the AUG start codon, and this purine-tich region is called the Shine-Dalgarno sequence (e-. for Ecol acl, the sequence is '-AGGAGG-¥), This sequence pairs ‘with a complementary sequence very near the ¥end of the 16S FRNA of the 308 subunit, ‘Therefore, proein synthesis willbe initiated where the amicodom of the URNA, binds the initiator AUG codon, and where the mRNA pairs with the complementary sequence at the 3° end of the 16S ¢RNA moleeule, Initiation in eukaryotes ‘The mechanism of initiation of translation in eukaryotic cells is fundamentally the same as in prokaryotes, but 1 The initiating URNA is Met and not formylmethionine (Met). The URNA carrying Met is termed RNA, 2 There are many more IFs (atleast nine are known, and doubtless ‘more wil be discovered) (a) elF2 binds GTP and escons (RNA tothe 408 complex: (b) cap-binding proteins hind to the mRNA "cap, and efF3 binds to the AUG start codon nearest 1 the cap, using energy provided by IES, which in tum derives its energy from ATP: (c) Met-IRNA, binds to the start codon AUG and clF5 causes oF? to hydolyse GTP, which results inthe release of elF2 and elF3 from the initiation eomplea: (4) the 60S subur ccomples, th some ciferences. is attached to form the complete initiation ‘Nave: in eukaryotes, there is one start codon only — AUG — and ‘io Shine—Dalgam prine-cich sequence, The 408 complex attaches itself tthe mRNA atthe 5"end and uses energy (ATP see (2a) above) to move towards the ¥ end until it finds the AUG stan signal17 Protein synthesis III er aT | Ni naWA » > d soa viene | roan signal peptide \ particle Nhe Ny Fig INTRODUCTION After the initiation of protein synthesis, the peptide chain is el Signal from the ted jon is terminate. pmal apparatus and may be subject ‘and when the a ed, elon, oe Von Np 2h COOH ‘io 7) ® ang Brine! oy © OH OH OH “Es om — Poros > ° ~_ingysl ca f3 enna) gt ars Lemna? Toner eR | Sg Ne 200 =" Exced sal aa oni to posttranslational modification. The protein will be modified depending on whether itis destined for other cellular organelles, uch ‘as mitochondria, the nucleus or lysoso 8 the cell membrane. It may be nits, such translational modification may result in disease ,ELONGATION “The amino acid tobe added to the intial methionine i delivered, tached tolls tRNA, to the A site ofthe ribosome by an elongation factor. Its critically important that the correct aminoacyl-tRNA is inthe A site, because the cell eannot excise a “wrong” amino acid once it has been added tothe chain. The ‘proofreading’ is dane by an elongation actor, EF-TU in prokaryotes. EF-TU binds GTP, and this enables ito bind the aminoacyl-tRNA and bring itt the A site. The amino acid cannot ‘be added to the chain until EF-TU leaves the complex, and EF-TU cannot leave uni it has hydrolysed its GTP to GDP. Therefore, thee is ime, ‘while GTP is being hydrolysed, and while the EF-TU is leaving the noacylIRNA to leave the complex, In ccukaryores, the prooffeading elongation factor is EFI, whose subunit EFlo.forms a complex with the aminoacyl-iRNA, Elongation has wo main steps, peptide bond formation end translocation. Peptide bond formation is catalysed by peptidyl transferase, which ataches the carbonyl atom of the P ste aminoaey— ARNA to the «camino group ofthe amin acid ofthe aminocyl-4RNA, inthe A site. The A site must now he emptied for the next aminoaeyl RNA. and so the reading fame i shifted three bases along the mRNA, ‘until the next colon isa the site. This movement is driven by another clongation factor, or transloease, which utilizes GTP hydrolysis for ‘energy In prokaryotes, the factor is EF-G, and in eukaryotes its EF2. During the process the tRN/A-OH whose amino acid has been removed is shifted toa so-called “exit site’ on the ribosome. from where itis released back into the eytoplasm. In eukaryores, a number af other clongation factors have been identified but ther exact roles are not cleat complex, for an ineorrect ‘Termination Whea a termination or STOP codon is reached, a0 aminoacy!-IRNA canbe accepted in the A site, Instead, the cox is hound by a release factor-GTP complex RE-GITP. As result, peptidy tansFerase switches to boing a hydrolase, alding H,0 to the earbonyl end of the peptide chain, RF hydrolyses its GTP to GDP. and undergoes a conformational change. These changes provide the energy to dissociate the elongation complex into the constituent mRNA, polypeptide chain and the ribosomal suhunits. In prokaryotes, three release Factors have been described: RFI, RF2 and RE3. RFI and RF2 recognize diferent STOP «codons, and RES potentiates the ations of RPI and RF2. Prokaryote-eukaryote protein synthesis summary Feature Prokaryotes —— Eukaryotes Large ribosome subunit 0s Smal bosome sunt 308, Whole ribosome 708 [Lage subunit RNA 38 SS 588 288 Small subunit RNA 16s, ass [Large subunit protein aambers 4 proteins Si proteins Small subunit rosein umbers 21 proteins 4 proieins Initiation factors IBLF AIFZcIF3 clPa Fah as etFSe1F6 cap-indingprtsin Initiasing aminosey!ARNA —— fMetaRNA MetaRNA restart purinerich sequence Shin 8 Release factors Dalgarno None ERG (ranslocase) EFI EF2 (eanslocase) RFURFZRES RE Post-translational modifications ‘The amino acid sequence and conformational shape of a protein will determine its Tate, whether i isto be targeted to particalar site, oF to be substrate for modifying enzymes. They will also determine its hale, Eukaryote ribosomes that produce lysosomal proteins, membrane proteins and proteins for export are bound to the ER. During protein synthesisin the cytosol, a signal sequence rich in hydrophobic residues, such as phenylalanine, is produced near the amino terminus, The sequence is recognized by aribonucleoproein termed signal recognition particle (SRP), which binds to the ribosome, enabling ito bind to the surfuce of the ER at a “docking” protein called SRP receptor. The ribosome interlocks with two ER membrane translocation proteins called Riophorin I and 1, which drive the elongating peptide chain through the ER membrane into the lumen ofthe ER. Once inside the lumen, the signal sequence is excised, Tnsde the lamen ofthe ER, several modifications may occur: (i) the protein may be crosslinked by disulphide bonds; (i) part ofthe chain may be excised by proteolysis, for example the removal ofa portion of several prohormones, such as the conversion of proinsulin to insulin, for expor; and (i) proteins may be glycosylated. Glycosylation of proteins serves three main purposes: (i) changes their physical properties, forexample solubility, size and stability (i) the carbohydrate addition is an important component of 2 membrane protein which has {o recognize other proteins or cells; and (ii) enables the protein to be targeted to specific celular sites. Inthe lumen, oligosaccharides may be carried o the growing peptide chain by a lipid carrer, dolichol phosphate, which is located on the minal surface of the ER membrane. The oligosaccharide attached to the dolichol phosphate bocomes attached to an asparagine (Asn) residue ‘on the peptide, in a reaction catalysed by a glycosyltransferase. Glycosylation may take place after the protein has moved through the ER tothe Golgl apparatus, where it may also be packaged into vesicles for exovytosis if itis For expor. Proteins may be modified during translation (cotranslationally) through te alteration of amino acids. For example, in collagen syn thesis, proline is hydroxylated to hydroxyproline, TARGETING “To the mitochondrion Proteins targeted to specific cellular sites need special signals, Most proteins destined forthe mitochondrion are released into the cytoplasm by flee ribosomes, and their signal will differ depending on whether they are targeted tothe mitochondrial membranes oro the mitochondrial matrix. Once inside the mitochondrion, the targeting signal is usually » Af > xs, We) om je eas ai. As sit () Poot vans fos @ COOH teins othe poypepic,wrch dssocaos Lk Ai) IBNAard mANA dssocate tom the complex ae (i) Therose dstines rb 0 ne 80 srs, FW 50 and promt omar bg 30 ar 6. Peptide bond formation NH . mat | ng rong Ny elypeptise 3 Polpeptie : @ Peis tomation: is catayz0s owing Ses © Set eer som ‘UUA —miNAS polypeptica } 4 I—H forming part of the 50S unit é, = Ry- G-H hang oe Ae Translocation: is A Pepiidy! 0 + Or We tome eH Nie EAAGH on be Fe ¢-H——> GH Re-G—H Se hate Baia Gat iesoinone nos ‘Stato pay Q — Ndconpas te? aa ste ongaton face ercams fancaon vp Pry) BNA je . f= of WW aN omect 7 Elongation factor cycle | Sorgen at EF-TU irs TP an ‘EF-TU binds GTP, EF-TS cesocits ang EF-TU-GP ready tne ancner melee of n aminoacp NA Our coe waa © EF TUonttes pestogo! amon ANAIn Abosnal se rs GT sea GDP ble FU aves Po rheve EF-TU.GOP bins eengatonfacor EFS end GOP dssoits tom the complexPATHOPHYSIOLOGY OF PROTEIN MODIFICATION Several diseases and aboormal protein status situations arise from errors inthe post-translational modification of proteins, including errors in the targeting process. Familial hyperproinsulinaemia is an autosomal dominant state in ‘which the individual has approximately the same amount of proinsulin and insulin in the circulation, Those with the condition may exhibit none ofthe symptoms of diabetes, and glucose metabolism i apparently normal, despite the presence of very high levels of proinsulin in the ‘nlood. The eause of the condition is not known with certainty, but may involve poim mutations in the proinsulin molecule, which prevents the action of the protease enzymes which spice proinsulin cell disease is one of 8 number of related disorders arising from aberrations in the targeting of lysosomal enzymes, Other diseases ‘are mucolipidasis IT and HI The lesion isa deficiency inthe enzyme Which catalyses the transfer of N-acetylghucosamine phosphate 10 plysaocharide moieties of proteins which ae targeted tothe lysosome. ‘These proteins are secreted into the bloodstream, and are found in high ‘concentrations ina numberof body fluids, The defect is usually present at birth and manifested by skeletal abnormalities, facial coarsening of features and psychomotor retardation, Patients usually die before 7-8 years of age. ”18 Errors of translation eT RUS ay Caria natarnce ff mors of vaniaion ff wentvarelaton Hyparproirsuinaeia Homanerestance ff Colagen sts ntti ion actoral toxins Ch he Ne Fibosome x Me rong Au oH vente ee oe. yn bay / oy n / ie weno Saye geo 6 i cn Pte Puromycin ‘Aminoacyl-tRNA is biotics, This Protein synthesis an be blocked by drugs, termed ar They may interfere with cell wall synthesis oF with intermediary metabolism. They are useful tools for elucidsting the mechanisms tremely useful therapeutically because hey ifically. Defects nsible fr several diseases inthe wanslation roc in humans Agw [ie Premature ended paptde chan INHIBITORS OF PROTEIN SYNTHESIS Antibiotics, Streptomycin and eomyein are aminoglycoside antibiotics nhibits initiation by Jy used therapeutically. Streptomycin binding tothe 30S ribosomal subunit and interfering withthe Funetionin of single ribosomal protein called S12, which mediates the binding ofInhibitor ‘Site of fnibiton Process inibited Chloramphenicol Prokaryole SOS subunit Peptidy wansterse Cycloheximide ukaryote 80S ribosome Elongation Enthromyein Prokaryte SUS subunit Transoeation Fusiic aid Prokaryoe EF-G action Translocation Neomycine Prokaryotes many sites Translation Puromycin® Ribosome Peptide transfer Ricin ukaryote Many processes Steptomyein Prokaryore 30S subunit Iiiton Elongation “euneyclines Prokaryoe 308 subynit ——Aminoacyl-IRNA Binding + Eokayotes and prokaryous. {Met-aminoacyl-4RNA and mRNA to the ribosome, It also causes ‘misreading of the mRNA codons. Neomycin does not appear to act through S12. etracyetines also bind 10 the ribosome and block aminoacyl-4RNA binding. They were heavily prescribed in children until it was discovered that they stained their permanent teeth yellow. (Chloramphenicol is toxic to a wide variety of microorganisms, but is also seriously toxic to hone marrow, and is reserved for dangerous sliseases such as typhoid fever, or for localized (wopial) application in, for example eye drops Antibiotics, such as eyeloheximide and puromycin, which attack both prokaryotic and eukaryotic runslation, are vitally useless therapeutically but are very powerful experimental tools. Puromycin resembles the terminal aminoacyl-adenosine pao the aminoacyl RNA and therefore competes with it for the binding site on the A site of the ribosome. PPuromycin becomes incorporated into the growing peptide chain atthe carboxyl end because it has an c-amino group recognized by peptidyl transferaye. Reins a plant woxin from eastor beans, that has N-slyeosidase sctivty and attacks the ribosome directly, cleaving an adenine base from the large subunit. Recently. in Spain, many hundreds of people died after using cooking oil contaminated with ici, “Translation can also be inhibited by bacterial toxins, For example, a prowin toxin produced by the diphtheria-producing bacterium ‘Corynehacterium diphihera, enzymatically inactivates EF2 by converting itto ADP-ribosyl El TRANSLATION AND DISEASE Several usually familial, diseases are caused by defects in the translation process, These may involve alterations to the cleavage of newly synthesized peptide chains, or defects in the targeting of proteins or from mutations in the genes, and thus in the mRNA, resulting inthe {incorporation of the weong amino acid into the protein. Cleavage Defects of cleavage ute responsible for familial hyperproinsulinaemia, {in which affected individuals have abnormally high circulating levels of the insulin precursor proinsulin. Normally the insulin gene eodes for a lange precursor. preproinsulin, which is released into the lumen of the ER. The signal sequences cleaved by a signal peptidase to yield a smaller protein, proinsulin. Tiss transported to the Golgi apparatus, where itis packaged into vesicles for export. Inside the vesicle, the molecule is ‘leaved further to remove the so-called C peptide, an intemal peptide ‘chain, and bioactive insulin is eventually released by exceytosis, Receptor defects and hormone resistance Errors of translation ean result n hormone resistance. This is failure ofthe target cell to respond to certain hormones, for example androgens, glucocorticoids, thyroid hormones and vitamin D, which ll act through Intracellular receptors In patients with androgen resistance, these receptors may be absent altogether: (i) grossly altered in structure due to deletion of segments of the carboxyterminal end of the polypeptide chain: oF (ii) altered in just one amino acid (a point mutation, which results from a mutation ofa single base pair in the DNA). All these are due to mutations of the androgen receptor gene, and the condition is therefore familial. Asa result, the patient does not respond to his oxen androgen, sand is infertile ‘Motations of ciferent regions ofthe low-density lipoprotein (LDL) receptor gene can cause a diseuption of cholesterol metabolism, and lead to hypercholesterolaemia and premature vascular disease. ‘The ‘mutation may result in: (i) areduced number of receptors or none at all: i a redueed rate of transport ofthe newly synthesized receptor from the ER to the Golgi apparatus: (i) the failure of the LDL receptor to bind LDL; or iv) failure of recycling of LDL receptors. These diseases ‘ae usually Familial ‘There are defects of translation of the proteins of collagen, resulting in structural weaknesses of collagen, an important supporting tissue in the body. »Fie wa 19 Collagen Col Collagen is used i tis chapter as an example of the evens in protein synthesis deserted in previous chapters. NATURE OF COLLAGEN Occurrence and structure Collagen isa fibrous, secretory protein, the most abundant protein in the human body and occurs ial tissues that demand a framework oF suppor in order o give them structural strength and retention of their characteristic shape o¢ form. Other examples of fibrous proteins are ‘uopomyosin and o-keratin, Structurally, fibrous proteins possess ahigh proportion of regular secondary structure and a rod-like cylindrical shape, and are relatively insoluble in H,O. Each collagen peptide, referred 10 as an ee-chain, occurs as a left-handed helical polypeptide, in which every third residue is a 0 eS eS slycine, and is about 1000 residues long, Three @-chains intertwine to form a right-handed tciple helix, and the glycine residues are ot the cente ofthe helix. This helical structure is termed tropocoll and it is the fundamental building block or repeating wnit of collagen, ‘Tropocollagen “Tropocollagen can be expressed in terms of an approximate Structural formula: (Gly-A-B),., where proline occupies approximately one-thint of the positions, an about one-third of the B postions are occupied by hydroxyproline, Tropocollagen molecules spontaneously combine through a erosslinking between Iysine and hydroxylysine residues. form the so-called polymeric fibrils, which can be seen under the electron microscope, and these aggregate to form the light microseope-visible polymeric collagen fibres.‘The rigity of collagen is due wo the presence of proline residues. ‘The strength of collagen is provided by the fact that tropocollagen molecules are adjacent to each other along approximately 75% of the length of each molecule. Because of the way they ate attached, they also resist stretching, o tensing. and will rupture if subjected w excessive tensile forces “Types of collagen Different issues possess different types of collagen, of which there are least 13, ealled type 1, I, et., up to XML Collagen types difer in terms ofthe o-chains, which are termed atl snd 62. Type I the first to be characterized, is present in te lagest abundance in the human body. SYNTHESIS OF COLLAGEN Collagen is symhesized by several different specialized cell types, for ‘example by the osteoblasts in bone, fibroblasts inthe tendons and by ‘chondroblasts in cartilage. Synthesis may be thought of in terms of intracellular and extracellular evens Intracellular events In the cel, the frst step is the synthesis of an c-chain by the ER- attached ribosomes, which translate the @-chain sequence from the mRNA as well asthe propeptide sequences, and the N-terminal signal peptide, which directs the newly formed protein into the lumen of the ER. The propeptide sequences ensure that the precursors remain soluble, as they do not form a helix. In the lumen, the enzyme signal peptidase removes the signal sequence, and the pro-a-chain moves along the smooth ER and the Golgi apparatus towards the plasma membrane. During this voyage, -OH groups are added to proline and lysyl residues which use vitamin Casa cofactor. The sugar residues glucose and galactose are added t0 hydroxylysine; the extent of glycosylation Uetermining the thickness of the resultant collagen fibrils. The sugar residues reduce the degree of packing of the tropocollagen microfibrils into polymeric fibrils. The extent ofthe glycosylation depends on the tissue in which collagen is formed. During the late stages of glycosylation, three pro-c-chains are formed, {nto unity thiol (SH) bonds between the propeptides atthe C terminal and the linked chains coil into the characteristic triple helix of twopocollagen. The Golgi apparatus packages the soluble precursor (procollagen) into vesicles. which are secreted from the cell by pinocytosis, Extracellular events ‘Once outside the cell, the fibre-forming collagens (types 1, IL and Il) lose the propeptides by enzymatic cleavage to yield tropocollagen. (Type IV tropocollagen does not lose the propeptides.) These molecules spontaneously aggregate to form fibrils and as the polypeptide chains build up a network of erosslinkages, so the strength of the collagen fibres increases. PATHOPHYSIOLOGY Scurvy Scurvy i the resull ofa deficieney of vitamin C (ascorbic acid), whose lack inthe diet causes a decrease inthe synthesis of hydroxyproline. (Note, unlike many other animals, humans lack the enzymes required to synthesize ascorbie acid from glucose.) Hydroxyproline cont bbutes additional hydrogen bonding for stabilization of collagen hnlices. As a result, collagen loses it stability at body temperature, and structures cannot adhere to connective tissues. The consequences include suppression of growth in children, capillary fragility and delayed and deficient wound healing. Teeth become dislodged from the gums, ‘and sudden death can result in extreme cases if the patient changes posture. Other disorders ‘There are, in addition, a numberof genetic disorders that result in: 1 failure of collagen fibrils to crosslink, for example, osteogenesis Jmperfeeta, whee poi mutations in the a-chuin prevent helix formation, and enzymatic destruction of the tropocollagen molecules oecus: and 2 deficiency of certain enzymes, such as Iysylhydroxylase, which is called Ehlers-Danlos syndrome type VI. Here, synthesis of hyro- xylysine is suppressed. ‘These diseases are characterized by poor wound healing, multiple fractures and hyperextensible skin and joins. aFig, 20 20 Control of gene expression in prokaryotes een cap Noinoveer present Lacoperon Promcter Operator SES 33ers a oN ‘Repressor mANA ww ° | =S333= =) 3 0-88 Rceime Repent | shunts, | @ inaucers Inducer present NA paumease ae F-gascceiduce Prmease PROKARYOTES E, coli « bacterium in which gene expression has been extensively died and characterized. . coli has a single, circular chromosome, anded DNA molecule of about 4 x 10 hase pairs. There are shout 3000, composed of a double nes, which are clustered according to or enzymes of a particu ‘metabolic pathway are clustered, asare those coding fr structural proteins. These clustered genes axe usually under co-ordinate contol, and are transcribed tog o forma single strand of mRNA that codes for several ferent proteins. Such an mRN) and a complete set of functionally clustered strands termed polyestronie mRNA. nes, together with their nes, is termed an operon, latory genes cou for proteins that in kum control expression of ‘operator and re eee Lactose netaboiing naymes Low gucose canceraion incel Transacayae the gones by binding 10 control elements a sites on the DNA near 10 the structural gene. Regulatory proteins control the degree of access thatthe enzyme RNA polymerase has to is binding ste om th Two types of regulatory protein have been found: () negatively actin which repress the operon by binding to the operator: and (i) positively acting, which enhance the afinity af RNA poly. ne. A good example of an operon is te lactose operon of ase for ils bind f OPERON ated by an inducer (lactose), and by a expressed by the f ge Expression ofthe operon is repressor prote “The / gene (also called lact), i situated just before the controlling elements forthe clusterBenes coding forthe enzymes, t least two of which are important in ‘the splitng of the disaccharide lactose into galactose and glucose. These thee genes, called the facZ¥A cluster, code for: 2) Brgalactosidase, Which acts on kitose i) Begatactoside permease, x membrane-bound protein which forms part of the transport system for taking lactose into ‘the cel: and (i) Begalactosidetransacetylase, whase precise function is unknown, but whose expression is essential forthe metabolism of lactose. The mRNA transeribed by the lac operon is extremely unstable, having half-life of approximately 3 min, which means that expression of the operon ean change rapidly. As soon as inducer concentrations fall, expression of the gene ceases, ‘The lac repressor ‘The / gene codes for a repressor regulatory protein, called the lac repressor, and te ene itsels not regulated but continues to produce the repressor ata Jow level, independent of other cellular events. The Jac repressor is expressed as a monomer of 360 amino acids, which fare associated to form a tetramer, and there are usually about 10 tetramers in the cell at any one time. The tetramer binds with high affinity to a specific DNA sequence situated between the promoter called ZaeP, and the operator called JaeO, forthe z gene. This binding reaction blocks the binding of RNA polymerase tothe promoter. The ‘operon is said to be repressed, Derepression of the operon LLactove induces or derepresses the operon by binding to specific high- affinity sites onthe tetramer subunits. The binding reaction causes an allosteric change in the tetramer, which drastically lowers its affinity forthe DNA sequence to which it usually binds, Lactose is not the only inducer. A number of so-called gratuitous inducers have been found, including sopropylthiogalactose, which bind to the tetramer but are not themselves metabolized by Begalactosidase, ‘an are therefore useful in the study ofthe fac operon. The mechanism deseribed above is an example of negative control. But, lactose ‘metabolism can also be under positive control. POSITIVE CONTROL E.coli prefers glucone to lactose asan energy substateif theres plenty ‘of glucose inthe cel, the lac operon is repressed, even if there is plenty ‘of lactose present. This is known as eatabolite repression, since it happens only when glucose is being metabolized. ‘The cell will turn to lactose as a substrate only when glucose ‘concentrations fall. When concentrations of glucose are high, those ‘of the second messenger cAMP are low. When glucose concentrations, fall, concentrations of AMP in the cell rise, and cAMP binds to a protein called eatabolite activator protein (CAP), which is an allosteric protein. CAP undergoes a conformational change as a result of the binding reaction. This enables CAP to bind to the promoter just before the RNA polymerase binding site, and this in turn facilitates the binding of RNA polymerase tothe promoter. CAP synthesis is regulated by a gene that is not a component of the ae operon. CAP is an activator of a number of other genes. including the galactose and arabinose operons, snd is probably a eo- the general control of enzyme symhesis whose activity is unwanted ‘when high concentrations of the preferred energy substrate (glucose) are high. ator for21 Control of gene expression in eukaryotes Oe ea oes) $= Splicing choice sis SA st ym Ss enn ® Pinay tansret |-—E—= amr —t + @ 3 @ Sen 2 @ @ mena an E 9 Pipa mine @ ae ee @ Tom eT ie of rasaton Caleonin ‘cae (CGRP = Calcio gelato peptide ‘Tropomyosin | gene of Drosophila fruit fy) Na Fis 2141 COMPONENTS OF GENE CONTROL Levels of regulation and Control is effected at three levels: (i) nuclear RNA synthesis: mRNA Gen santrol has three main components: (i) signal: (i) and (i) differemial prooessing of primary transcrips: an (ii alter ‘iro of nuclear RN: fected ch as heat shock stability inthe eytop ‘mainly atthe initiation stag factors (activators), which may int RNA polymerase Il ‘with genomic prom an mRNCornus Levels of contot Exyropostin hick Oestogen —] Nuclear RNA synthesis Mech jams of tensions conta D— Trrscrpona tas i (QA DNA unig Calor organism Transcription factor Mammalian lls Spi Promotes con bores. eg. aitydofolae reductase promoter or CAAT box Drosoptila ———B protein ‘TATA box STE Promoter of heat shock ‘nes Biympboestes Immunoglobulin enhancer Enhancer ssquence for expression (CTE CCAAT-inding wanscrption factor; HST, heat shock wamcrption factor Differential nuclear processing of transcripts. Differential cl eof polyA sites on the primary transcripts determines tissue specificity of ‘gene expression. For example, in ral, the transcript encodin hormone ealeitonin also codes for a brain peptide, caleitonin ge related peptide (CGRP), and in the thyroid gland the cells involved proxiuce calcitonin, while inthe brain CGRP is produced, Cell or organism Transcription factor Genomic target Ghwcoconicoid- — Glucooortsoid receptor $-GGTACAmATGTICT" responsive consensus sequence cel {namy nucleotide) genes coding fr ©. chicken Yeast 38 protein Specific upstcam sequences in promoters of genes expressing metabolizing enzymes Cstoplasmie gene control. The rate of protein synthesis may be afected by: (ithe rate of transport of mRNA into the eytosplasm; (i) hall-ife fof mRNA; (iil) frequency of mRNA translation; and (iv) post- translational cootrol. Control of halflife of mRNA may’ be enhanced by hormone, the occurrence of DNA replication, tissue (liver) and by certain viral proteins,22 Mechanisms of transcriptional control eMac Tu Rit Control of $V40 early RNA synthesis DNA sythesroion =—_— T antigen ties teh gh afin ost T w~ rage par varscipt aes ‘ernigen More arigen moos a ee i Tntge io ste art wt have aloe any | Tontgen ard vansrpion of w~ ‘T anagen ANA inhibited Trreciion of Tantgen cary nay rsp Doce Pig 2 e INTRODUCTION This is a brief, but more detailed look at some of the mechanisms underlying used are the following, 1 The role of the T antigen inthe control of transcription inthe SV40 Virus, which is papovavinws. These area family of smal, non-envelopedt double-stranded DNA viruses, including the papilloma viruses which produce the common wart, some of which can be oncogenic processes whereby transcription is controlled. Examples 77 Macey MMTV rowpotes ino hast ONA 2 Te MMTV role of glucocorticoids in the conttol of transcription of the 'Sv40 VIRUS. When SV40 virus infects cells, its DNA is transeribed by host RNA, polymerase Il, SV40 DNA contains two transcription units: one termed ‘arly’, and the other termed ‘late’. This is because the early unit is oially transcribe fer infection soon wl the late unit ispreferentially transcribed later during infection. The shift from early to late stages is mediated by a protein called T antigen, which is produced during early transcription. T antigen, which functions as a tetramer, has three binding sites on SV40 DNA: I. {land IL Binding to may increase the affinity of T antigen for sites Hand IIL, thus inhibiting further transcription of the early primary transcript ‘T antigen may therefore be an autoregulatory protein, which inhibits the synthesis ofits own mRNA. T antigen was the first ofthe proteins to be discovered in eukaryotes that binds to specific sites on the DNA. MMTV ‘Steroids are lipophilic substances which pass easily through the plasma membrane and combine with specific receptor proteins. These ‘complexes bind to specific sites on the DNA to alter transcription For example, glucocorticoids are substances that bind to intracellular receptors, Glucocorticoids are known to stimulate the production of increased numbers of MMTV molecules in cells infected with the ‘Note: retroviruses are enveloped, single-stranded RNA viruses, {including human immunodeficiency virus (HIV). which infect cells, fare converted into DNA by the enzyme reverse transcriptase. The DNA is incorporated into a host chromosome and may preferentially be transcribed back into the virus or remain dormant for several ce generations. a723 Growth : Signal transduction and cell growth ees Flxotast row aco ‘hou ‘Grow eto GF) + Receptor I ; z ne oo Hours Pratoeterned grow acer oF Fer oF Fens Hint . eee coewn| _ SFRns = eke spay \ © in govn eda foasecate Cell cycle in mammalian cel (e.g. 16 hours) PP a convo acti rama | none recta US Wun sep ys cee ) [plse == EO fom a) Gan) MSO ULE Guns) ear seseste ‘STAT signal transducer 4 | ree i [Leromn ] “TF Transcription tactor < ng ‘Signal transduction vue erie even ee are rns ae see in duration (7,3 and 1h respectively). During mitosis, RNA synthesis The eukaryotic cell has life eycle characterized by fou ‘ceases and protein synthesis is greatly reduced, successive phases, called G,, S, G, and M. G stands fo “Mitosis is typical process of nuclear division, when two daughter synthesis and M for mitosis. DNA is symhesized during the § phase, nuclei are formed, each having the same chromesomal complement as ‘while RNA and protein are synthesized during G,.S and G,.The .G, the parent nucleus. Cell division occurs after a full eyele has beenGrowth factor oF hormone® Main sources Mouse submaxillary gland Embryonic and cancerous ells GF Transforming growth acer a (TGF-«) 13-8 Mont cells cancer cells Exytropociin Kidney Insolin Pancreatic [Neve growth fator (NGF) Many cells POG Patel IGF-I: somatomedin A Liver cH Anterioepititary Target tissue or ell Epidermal eels Grobe 2 bros fe fibrobasis Red lod eel precursors (entvablasts) Liver muscle Sympabetic nerves “Arterial smooth muscle els ropa faneton) Mediates GH action on growing bone: mitogenic in sme cells Liver-timlates somaomedin prodicton “Tiss is moe comprehensive. completed, Some cells, such as muscle and nerve, may never divide lier formation, while others, such a liver, skin or gut, will be active in cell division, When the cell enters.a phase called G,, it is dormant until trigger (usually a growth factor) switees it nto active cell division Certain growth factors, notably platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGE), and insulin-like growth factor (IGF-1) and IGF-2 (see below), stimulate the cell eycle at various points during G, GROWTH FACTORS Growth factors are peptides that stimulate cellular proliferation, They regulate normal growth and development. They may also cause what i ‘known as phenotypic tansformation of cells, which may result in cancer. They produce these effets through an interaction witha specifi receptor fn the cell surface. They fall into several arbitrarily classified groups, 1 Hormones, such as insulin, secreted by the B cells of the pancreas, growth hormone (GH) and prolaetin, bth of which are secreted by the anterior pituitary gland, 2 Cytokines, which are not necessarily hormones, and which are made bby many different cells. They affect growth and division of eels 3 Lymphokines, which are polypeptides released by activated ‘macrophages and cells ofthe immune system. Some influence white cell migration, while others also act as growth faetors. Interleukin (IL-1), which is released by macrophages, stimulates proliferation and differentiation of the B lymphocytes. Mechaniesm of action of growth factors Grow factors exert their effects on cells through membrane receptors. ‘When the factor binds to the receptor the signal is transferred to the cell in the form of a cascade of several reactions, especially phosphorylation reactions. Tn certain eases, the intracellular domain of the receptor contains a protein kinase that enables the receptor to aulophosphorylate itself (see below). The final result is initiation of transcription, protein synthesis and growth, ‘The growth factor receptors can be intracellular phosphorylation cascade assed in terms of the Insulin receptor substrate 1 (1RS-1) signalling system. Some hormones, and growth factors, for example insulin and IGF-I, bind to receptors that contain intrinsic tyrosine kinase activity and avtophosphorylate themselves. This results in the tyrasine phosphorylation of a protein called IRS-1 This enables a group of so-called SH2 proteins (proteins with sre homology) to “dock’ at IRS-1, resulting in conseque intracellular events culminating in the mitogenic response. Some growth factors, ©, I. bind 1 a receptor that does not contain intrinsic kinase activity, but which recruits a cytoplasmic tyrosine kinase that ‘hosphorykates IRS-1 on tyrosine to create the SH2 binding sites, ‘Note: the insulin receptor consists of two c-chains, each of which binds a molecule of insulin, and two B-chains., which span the ‘membrane and contain the catalytic sites, The chains are held together by disulphide bonds, Receptor kinaseSH2 system, Certain growih factors, for example EGF and PDGE. bind to receptors that possess intrinsic kinase activity, which allows them to become autophosphorylated, and these phosphorylated sites become docking sites for SH2 protein ©Mtokine receptor superfamily (CRS). This is @ group of hormone and arowth factor receptors that do not themselves have any kinase activity, but which activate a group of eytoplasmie protein kinase kinases, called the Janus kinase (JAK) family. Atleast four members ofthe JAK fanily have been identitied: JAKI, JAK2, JAK3 and TYK2, and there are probably more. The JAK proteins bind tothe membrane proximal area of the setivated receptor, become tyrosine phosphorylated and in tun they hosphorylte the receptor causing the activation of the following. 1A group of cytoplasmic proteins that form part of a family of transcription activators, called signal transducers and transcription activators (STATS). 2 The phosphorylated STATS activate transcription atthe level ofthe DNA—the Ras pathway. The phosphorylated receptor provides binding sites for a group of SH2 docking proteins, which activates the Ras GTP system, resulting in activation of a group of mitogen-activated protein kinase (MAPK), and a transcription activator TE ‘Termination of action Growth factor action wil he terminated by the dissociation ofthe factor from its binding site on the receptor. It may also be terminated by the inactivation of the intracellular MAPK. There is evidence that the MAPK, may be down-regulated by dephosphorylating enzyme phosphatases, Furthermore it seems that when MAPK are activated this triggers the cexpressionof the genesencoding the phosphatases, which would provide tightly and elegantly controled mechanisin forthe regulation of growth factor activity. Any disturbance ofthis contr! system might conceivably contribute tothe factors that cause cancer through unrestrained growth factor action,Pig. a 24 Cancer Cancers the uncontrolled apparently autonomous grow of cells and their invasion of the rest ofthe body. Such cols are termed malign and their invasion is termed metastasis, Normal cells hecome trans- formed through mutagenie agents 50 MALIGNANCY Knowledge of the molecular basis of cancer stems largely froan the study of viral activation of oncogenes. Cancer-producing viruses Certain viruses transform healthy cells to malignaney. Viruses areinfectious DNA or RNA, which may be single or double stranded, surrounded by a protective coat consisting ofa large numberof repeating protein subunits ‘Viruses must invade other cells to multiply, and acid instructs the host (permissive) cello synthesize more viral nucleic acid protein. Tis invasion usually kills the cell, and dhe complete virion, oF virus particle, is released. Occasionally the cel is non: permissive, and may destroy the invading virus, or incorporate the viral genetic information int its own genome. ‘Some viruses that contin RNA are termed retroviruses. They have a polymerase enzyme tenned reverse transcriptase, which, when they have ‘entereda cell produces adouble-sransed DNA copy whichis incorporated imo bost DNA. The cells ransformed ether because the veal DNA copy ‘contains the oncogene, or because the viral gees atvate a host oncogene The avian sarcoma retrovirus binds to specifie cell membrane receptors, and inserts its contents into the cell. Reverse teanseriptase produces a DNA copy which is longer than the viral RNA template because it contains long terminal repeats (LTR), The LTR contains the enhancers, polyadenylation sites and promoters. The viral DNA cireularizes enters the host nucleus, and ig spliced into the host DNA, av TCAG sites ‘The 10kbp of avian sarcoma virus contains four genes, three of Which are necessary for infection. These are gig, pot and env. The other, se. is necessary for transformation to occur. Thee viral mRNA species are expressed: one unspliced mRNA coding For both gag and pot. and two unspliced mRNAs encoding env and sve sre product isa tyrosine kinase, The viral proteins and the unspliced primary RNA, ‘anscript migrate to the cell membrane and are incorporated into it Prt ofthe altered membrane buds off a new vial particle. Retroviruses «do not usually kill the host. The HIV is a retrovirus Oncogencs Oncogenes are eancer-producing genes. They may be present on the Viral genome, imported into the host genome or already present as a cellular gene which is activated through viral infection, Cellular ‘oncogenes (also called proto-oncogenes) are usually silent, or expressed normally under cellular control, But, iFa viral DNA copy is spliced into the cellular genome adjacent to a proto-oncogene, the viral LTR may stimulate its expression, thereby'transforming the cell ‘Oncogenes are named after the species und disease produced by the Fig,25 Genetic manipulation | Fie 2s : are amplified and recovered for further use. The genes im INTRODUCTION the host may cause it to express the gene products directed by the implanted gene. The vector may be a bacterial plasmid, a bactrioph fr the DNA may be introduced directly into mammalian isthe alteration of DNA, which is transmitted in ino host organisms, in which the manipulated gene(s) Genetic manipultissues using surgical techniques. “Techniques involve the introduction of the chosen DNA nucleotide ‘sequence into a vector the introduction othe vector into a hostin whieh the sequence will be amplified, the cloning of the altered host and the recovery of the amplified sequence. CREATING THE DNA SEQUENCE DNA from mRNA ‘The production of a mixture of €DNA from small quantities of mRNA, is made possible using the enzyme reverse transcriptase, obtained {rom retroviruses. Double-stranded DNA for insertion into vector can be prepared from the mRNA expressed by the gene. The procedure involves the following 1 The mRNA is isolated and given an oligo (A)T primer, after which it is incubated with reverse transcriptase and the four deoxynucleotides tomake the frst CDNA strand, 2 The first strand as at its ¥ end a hairpin bend whichis used, together ‘with a primer called the Klenow fragment and DNA polymerase 10 ‘make a second DNA strand complementary to the first, ater which the bend is removed with an enzyme, $1 nuclease, 3 The 3-terminal ends of the DNA are given homopolymer tacts of CCC....et., which combine non-covalently with tracts of GGG... ete, added to ends of a cut plasmid. After the plasmid with ts new addition ‘of the sequence of interest is annealed, it is introduced into the host tis possible to amplify the gene starting with minutely small, ‘quantities of mRNA. A disadvantage is that many thousands of different DNA molecules wil be cloned. Restriction endonucleases Cutting DNA at selected sites is possible through the use of restriction tendoncleases. The physiological role of these enzymes isto destroy ‘unwanted cellular DNA. Once DNA has been extracted, it can be cut ‘with enrymes that recognize certain sequences, and which cut the tracts inpredictable places. The enzymes share in common the ability to read palindromic sequences. (A pure palindrome.c.g. AATTAA. i identical ‘when read from left to right or from right 0 left) Some enzymes (eg. Alul) cut the DNA leaving “blunt ends, Ths is not ideal, since a ‘meeting between ends to be joined depends purely on chance. Some enzymes (eg. EcoRI) make sticky" ens, with sequences extending from the chin, allowing complementary base pairing with another chain, Fig. ce SEPARATION OF DNA FRAGMENTS Restriction endonucleases will eu the DNA into sevoral fragments of differing sizes. These are separated by gel electrophoresis. The negatively charged DNA fragments migrate to the positive electrodes, with the smallest moving fastest In oder to estimate the sizes, separate Jane is run containing Standard DNA fragments of known size. After electrophoresis, the bunds of separated DNA are visualized by staining the gel with ethidium bromide, which intecalates in the DNA and fuoresces under UV light. The gel can be photographed. This enables the estimation of the number of fragments cut. As litle as 25 ng of DNA can be scen this way. Ifthe starting DNA material is radioactively labelled with phosphorus-32.(%P), 1-2 ng of DNA can be visualized using X-ray film, The sie ofthe band is obtained by comparison with the migration rate of the standards. ‘Once the size and numberof fragments are known, andthe sites where they have been cut, its possible to star constructing a map ofthe DNA ‘molecule — so-called gene maps. SOUTHERN BLOTTING AND GENE PROBING ‘The technique of bloting involves transferring the separated DNA bands from the gel toa nitrocellulose oF nylon sheet, when the DNA bands are transferred by capillary action, Once on the shee. the DNA is fixed ‘wit by heating or chemical reaction, and the bands can be probed using a radioactively labelled gene probe. whose DNA sequence is complementary to that ofthe gene to be probed for. The probe hybridizes tothe gene, the unbound probe is washed away and the hybridized probe visualized. The experiment confirms thata gene has indeed been isolated, “The DNA of interes can be recovered and inserted into vector. RNA can be electrophoresed instead of DNA, and wansferted 10 8 nitroceltulose or nylon sheet (Northern blotting) the separated bands of RNA are probed with labelled cDNA complementary tthe mRNA expressed. This confirms thata particular gene has been expressed, and that ils mRNA is among the species of mRNA electrophoresed, Similarly, proteins can be transferred and probed using immunocyto- chemical techniques, when the process is termed Western blotting. POLYMERASE CHAIN REACTION (PCR) PCR enables us to amplify very rapidly a DNA sequence in a small biological sample. This allows the identification of potential disease Producing genes ina prenatal sample, the forensic identification of DNA for legal purposes and the amplification of sequences for insertion into vectors or for sequencing purposes. Principle ‘The method! was made possible by the discovery of organisms living near boiling geysers. Their enzymes, including DNA polymerase, ‘operate at high temperatures. PCR depends on the use of this DNA polymerase, a supply of the four () nucleotides and primers which lank the DNA tobe amplified, afer it has been denatured to give single the DNA is denatured at 90°C: (i the primers anneal to the sequence to be amplified at 50°C; (ii) the primer sequences are extended at 70°C; (iv) the eycle is repeated several times; and (¥) the amplified DNA is recovered from the eaction mix. Theoretically, iis possible to create over 250 million copies alter 30 cycles, ss26 Genetic manipulation Il Coir Sect done and araly gene To make @ genomic brary eces Popa teal genome DNA om engansm TR pee NS Sea eer BOs, tte agar Prbe lor pace genes wth Cone in col ard plat et iaeled pros Digest wih restnction endonucleases Se e ie ichecnaell | + O00000000 } ty sia E i] traci DA om co / Cel eontan O° DNA sequencing Her eaion Si Dengrucokie ssacmicined sae Separate using ecrephowes c fe oe 5A HOW ao er TT aes ! eat TT oman ae Ppp. ecoptores o newly sythaszad Counters \aeled cDNA acts a psence TGACATG-5 Sesonraceoee * eer wrabeewn dATP | ddcTe — a n° eoxyruceot des 1 _ “Mi Bi oo a i — | cr A (ss ucmaiie ~ACTOTA| —-ACTOTAC _- -ACT@ io adore aatte | ee i - -AcTG TA 7 -acta “act - -ACTGTAG 6 @ va ZAcTaT et © vectors host cells easily, readily confers selected phenotype character istics on host sand possesses single sites for many restriction A vector must be able to enter a cell, be sta within the cel. The idea ind able to replicate endonucleases, ‘vector has low molecular weight, encersCall by ‘antici von — Dacterophages: coms sree nage nen ew batt e ‘Medium cortans | svete ‘ony cortang ed das rend — ‘easier ene decane sean caren ‘em ages ms Plasmids (pBR322, Plasmid pBRI22 isan artificial citcular DNA molecule Plasmids are small circular DNA, occurring naturally in bacteria Inside bacteria, plasmids replicate independently of the chromosomal DNA. Plasmids enter bacterial cells more readily if the cells are first rated with CaCl, a low temperatures, or expose to an electric eld (leetoporaton’), CaCl, causes the plasmid t bind tthe cell membrane, and if the cell heated briefly «0 40°C. the plasinid rapidly eters the cell Transformed cel, those containing foreign DNA. will replicate in culture to form a done of daughter cells. Notall the bacteria in a culture ‘will take up the plasmid dat contains the gene of imerest; these have to be distinguished and separated from those that have taken up the gene Pasi, such as pBR322, have been designed to achieve this separation containing 4363 hase pairs, and contains sequences which ae the genes Which confer resistance to ampicillin and tetracycline (genes Ay/* and Te', respectively). The plasmid also has several sites for cleavage by restriction endonucleases, When the plasmid is cleaved, the sizeof every fragment proiced ean he calculated, Other vectors, Bacteriophages are viruses that infect bacteria, The phage binds to the bacterial membrane and injets is nuclei acid ito the cell where iis replicated. The phage can be “primed” withthe recombinant DNA one ‘wants to infect the ell with, ss Fig 6227 pH and buffers | DISSOCIATION OF H,0 ‘The dissociation of H,0 is through a breakage of an -OH bond: HO-H+ On o 25°C: [11] (081) = 10° mov @ ‘where [H'] and [OH] are the concentrations of the two fons in moles Per litre. The relative molecular mass of H,O is 18; therefore, 1 mol ‘of H.O weighs 18 g. One lite of H,O weighs 1000 g. Therefore, H,O is 1000/18 = $6 mol/. In pure H,O, [H"] = (OH}} = 10° mol pH is more convenient 1 refer othe hydrogen ion concentration in whole ‘numbers. To do this, log of [H*] is used (logis called ‘p'), Thus: pH =-log (Ht) eo ‘Therefore, when (H'| = [OH] the pH = 7... the neutral pH. Below 7, solutions are acidic, and above 7, solutions are basic (alkaline). Note that a change of 1 pH unit means 2 10-fold change in [H"]. In the body ‘the pH varies, depending on the function of the compartment. In the ‘ell cytoplasm the pH is 72. Inthe stomach, where food is digested by ‘acid, the pH is about 1. In the small intestine itis about 8, and in lysosomes it is about 5. ACIDS AND BASES ‘An acid is any molecule that can release a proton, and gains a negative ‘charge, and a base isa substance that can accept a proton, and gain 3 positive charge. Negatively charged molecules are called anions, and positively charged molecules ae called cations. Strong acids are acids ‘that readily lose protons and are 100% dissociated, and strong bases ‘are those tha take ther up, In an acidic solution, « weak acid (such as the -COOH group of ‘amino acids or the phosphate groups of nucleic acids) tends to hold ‘onto its protons (.e. remain largely unionized), while a weak base will take up protons (i. ionize). Ina basic solution, weak acids will ionize, hile weak buses ae only partially ionized, [Nucleic acids ionize by releasing protons, and become negatively ‘charged, Amino acids contain both basic (-NH,) and acidic (COOH) ‘groups. and can become positively or negatively charged, of both, Tonization of amino acids ‘Amino acids have “COOH groups which can release protons, and they have -NH, groups which can accept protons. Therefore, depending on the pH of the solution, amino acids can exist as weak acids oF bass. Since proteins may have unequally balanced numbers of COOH, and -NH, groups, changes in pH of the solution in which they are dissolved will cause changes in the ratio of charged acidic and basic ‘groups. For different amino acids, the pH at which the -COOH and se -NH, groups exactly balance each other to create no net charge on the ‘molecule is called the isoelectric pH ofthe molecule. In chemical tems, ‘any molecule that has both negatively and positively charged groups is called a zwitterion, ‘The Henderson-Hasselbalch equation, “The -COOH group ionizes thus COOH COO +1 0 At equilibrium: [coo 11H") (coon sil where K (also sometimes called Ka) isthe equilibrium constant for Equation (5). pK is defined asthe pH at which 50% of the ~COOH (or -NH,) groups are ionized. Clearly, K will depend on the numbers of these groups thatthe amino acid has. Similarly, the -NH,* group is deprotonated thus: N H+ He o Figure 27.1 is a titration curve for the -COOH and -NH, groups, showing that deprotonation of the groups occurs over a pH range, and the pH at which $05 of the groups are deprotonated is the pK. Note that at pH values below the pk. the protonated form predominates; at ‘lH values above the pK, the deprotonated forms predominate follows that a stronger acid has a lower pK, i. it reaily loses protons. Using Equation (6), we can derive one which enables us to predict the slate of ionization of a given aming acid if we know K andthe pH of the solution 1 Rearrange and take the log of both sides {coo} log = log [11'1+ log LOOT. 08K = log (H'I+ 108 Sony ® 2 Convert to -log and rearrange {coo} log [H'] = —log 1K] + lo log IH] = ~log IK 1+ 108 So o3 Express in terms of p(-log): the Henderson-Hasselbalch equation Ete arene fi cute dress Te val guy of ag fe apn tous eg eta ea eco eee rere gee err Shurtctpectiniane t eeaaitocanaed predicted BUFFERS Buffer solutions are those that resist a change in pH even when H° fons are added to, or removed from the solution. Thus, they protect the solutes within the buffer from sharp changes in pH that could, for ‘example, inhibit a chemical reaction. In the absence of a buffering ‘mechanism, the pl¥ of a solution will change much more when acids ‘or alkalis are added to the solution. Mechanism of buffer action ‘The weak acid, acetic acid (CH,COOH) (found in vinegar and bad ‘wine), and its salt sodium acetate (CH, COONa) provide an example of a buffering system. The acid has a pK of 4.75. A change of 2 pH units in the solution from 5.75 to 3.75 causes a change from about 10% CH,COOH in the unionized form to about 90% unionized CH,COOH. The ability of a weak acid and its salt to buffer a solution is greatest over the pH range pK -I to pK +1. When CH,COOH and CH,COONa are present together in solution, they ionize as follows: CH,COOH = CH,COO +H ap CH,COONa = CH,COO' + Nav «a Although the acid ionizes only paral, salts ionize virtually completely. ‘Therefore, there will be a large concentration of CH,COO- and Nav jons in solution. The increased concentration of CH,COO” ions from the salt suppresses even further the ionization of CH,COOH. If more FH fons are added to the solution, they will combine with CH,COO {ons to form even more of the largely undissociated CH,COOH. A new equilibrium is established, and the resulting liberation of HY ions is relatively slight IFOH-ions are added, they will combine with Hons to form neutral H,0. Ths, at pH values close to its pK. « weak acid isa useful buffering agent when mixed with its salt. ‘The system wil lose its buffering capacity sharply at pH values more than 1 pH value away from the pK. In a strongly basic solution the ‘weak acid itself ionizes virtually completely, so cannot exist in the ‘unionized form, and in strongly acidic solutions it cannot exist inthe ionized form. In cells, buffering of physiological fluids is achieved largely through the ionization of phosphoric acid (H,PO,), to form phosphates. H,PO, ‘can exist in thee forms, depending on the pH of the solution pk=21 HPO, HPO, +H 3) pK=7.2 HPO, HPO +H aay pK=127 HPO = Po, +H as) Ateytoplasmic pH, phosphat cam act as buffering systems, 728 pH and buffers II Buffers and aci Cee pH-HCO.” graph HOO; mgt » These ports were satained ug the 10 Headason-Hasebah quston 0 7 72 7A 78 78 Effect of concentration on the buffering line of blood «0 * PHYSIOLOGICAL BUFFER SYSTEMS ‘The major uid compartments in the body are the intracellular Maid (ICE), and the extracellular fluid (ECE), which consist ofthe plas und the intrsial flu, All are bounded by semmi-permeble membranes, ‘whose properties depend on ther function. All need buffering systems, which depend on the major ions of the compartment, Plasma buffering system In plasma and interstitial fui, the CO-bicarbonate (HCO, system is ent of dangerous acid or base very important, It prevents the develop imbalance, and works as follows: pH-HCO;" graph Poop = EHO; conponsies . fer: Fo (a) eat aio and t 0 respratory aks ga Buterng inoofboed 0 pH imbalance | CO, + H,0: 1.00, a Carbonic acid (H,CO,) is a weak acid, and ionizes HCO, H+ HCO, H.C, ionizes so rapidly that for our purposes the reaction of importance cain be considered to be: CO, + H,0 =H'+HCO, a Recalling the Hendetson-Hasselbaleh equation H = pKa + log COL _ 4 " *ico,11H,01 The pH in blood is 7.4 (actually ranges from 7.35 to 7.45) the pKa forHCO, is 6.1, Therefore, from the previous spread, we know that this is abutlering system that operates over the pH range of about S-7. [HO] is taken as unity. The concentrations of gases in fluids such as plasma are expressed as partial pressures (e.g. the Peo, in plasma ranges from 45 106.1 KPa). To convert partial pressures into concentration terms, it is necessary to use a conversion factor. For CO,, the correction factor, at 37°C, is 023 mEagf per kPa, For our purposes let Peo, = 5.0 KPa From Equation 4) IHCO, | O18 i975) © Therefore, under these conditions [HCO, ] = 22.94 mEq/ ‘The reader may have noticed that HCO, is not, theoretically, a good buffering system above pH 7.1, Yet, i efficient at buffering plasma at pH valuesas high as 79. Thisis because the body eliminates CO, through respiration, In other words, unissociated weak acid is being eliminated, and so Equation (3) is driven to the left. This enhances the buffering capacity of the system, 1CO, built up inthe plasma and other tissues, the body would suffer «an acidosis, but CO, is lost through the lungs. The pH-HCO,- graph shows how HCO, ion changes with pH ata given Pco,. When Pco, changes, the graph sifs in line called the blood buffering line. The buffering action of HCO, is supplemented by protein and phosphates, hich assist 10 "mop up” H° ions as they are formed. The steeper the slope of the buffering line, the better the buffering action. The slope reflects the concentration of Hb in blood. Hibs an important buffering agent in blood through its ransporting of CO,, and its ionization. Hb removes about 60% of H* ions produced through normal CO, transport, Therefore, in disease states involving a depletion of Hb, the buffering capacity of the blood will be reduced. ACID-BASE BALANCE ‘The body generates acids through metabolism and respiration. The ‘major respiratory acid is CO,, and important metabolic acids ar laetic acid, and the ketoacids B-hydroxybutyric acid and acetoacetic acid Tn addition, acids may be taken in the form of drugs such as aspirin {acetylsalicylic acid). Inthe disease state diabetes mellitus (see below) ‘excess ketoacids ate produced. Acid-base imbalance ‘There are four main types of imbalance. 1 Respiratory acidosis. Here, CO, is retained, either because of hypoventilation or intrinsic lung disease interfering with gas exchange. For example, hypoventilation may result from depression of the respiratory centre by drugs, while intrinsic lung disease could include conditions such as chronic bronchitis. I the latter condition, mucosal thickening and airway plugging with mucus may lead to poor alveolar ‘ventilation and CO, retention with low arterial Po, values. 2 Respiratory alkalosis. This results from hyperventilation. CO, is blown off through the lungs too rapidly. and blood pH rises. ‘Hyperventilation may be caused by poisoning with acids such as aspirin, by fever or by anxiety 3 Metabolic acidosis. This may arise from ingestion of acids or substances metabolized to acids (e.g. methanol intoxication, when ‘methanol is oxidized to formic acid rom overproduction of endogenous acids sucha ketoacids in diabetes mellitus; rom failure to excrete non- volatile acids in certain types of renal disease, including acute and chronic renal failure: from loss of base (HCO, ) reserve, for example in ‘severe diarhoea: or from loss of alkaline upper gastrointestinal contents after surgery (e.g. fistula formation), 4 Metabolic alkalosis. This can occur through ingestion of bases such as sodium bicarbonate. It can also occur ifcertain diuretics are taken, Diuretics are drugs that promote the flow of urine ‘The body compensates for metabolic acidesis by hyperventilation 10 blow off CO, and by increased renal (kidney) excretion of H* and HCO, regeneration. The body compensates for metabolic alkalosis by hypoventilation and inereased excretion of bicarbonate through the kidneys, although hypoventilation i Hmited by the fallin arterial Po, ‘which would otherwise occur,29 Chemical reactions | CHEMICAL EQUILIBRIUM reversible chemical reaction ean be characterized thus: AtBec+D ao ‘where A and B are eactants, and C and D are products Initially, the ‘concentrations of Cand D are low, but a their concentrations build up, 0 the reaction slows down, until thee is no further net change in the concentrations of any of the chemicals. The reaction has reached a ‘chemical equilibrium forthe conditions i, temperature and pressure, "under which the eacton occurred, and the ratio of reactant and products is constant, defied by the equilibrium const (scum) = (ATIBI,, @ where (A] and [B] are the molar conesntrations of reactants nd [C} and [D] are the moar concentrations ofthe products, K,,is useful, since it defines the reaction system under given conditions but it does not tell whether the reaction is going to oceur ‘or ot For that, we nced to know the energy level at the iat ofthe reaction, andthe energy level ofthe systema the end of the rection, FREE ENERGY Free energy isthe energy available for a reaction, andthe relative free {energies im system atthe stat and end of a reaction will determine Whether a reaction will take place or not. nother words, itis the change in free energy (AG) that is important to measure, AG is defined by AG = AH-TAS 3 where A means the change, G is fee energy, H is heat energy (also called enthalpy) of the system, is the absolute temperature and Sis the entropy ofthe system (see below). A chemical reaction ean occur only if AG is negative. Equation (3) was derived from the laws of thermodynamics, which \were formulated to predict the directions of chemical reactions, There , but only somte need coneemn us here Laws of thermodynamics ‘The first law of thermodynamies states that energy is conserved in a chemical system, i.e. the total energy of a system and its surroundings isa constant. The frst law is in effect the conservation of energy. The energy may he converted from one form to another, for example from chemical bond energies to heat, and vive versa hut the total energy within the system is conserved, There is an ‘equation derived from the firs law: AE=E-E, o «0 ‘where ABs the energy change, is the energy ofthe system athe end of the reaction and E, isthe energy atthe star AE and AG are related by the equation AG=AE~Tas 6 ‘The first lw eannot predict i «reaction will cecur spontaneously For this, another law is summoned, namely the second law of thermodynamics, which states tha a reation can occur spontaneously only if there isa net increase in the sum of the entropies ofthe systern surroundings Entropy is term that means, quite simply, the degree of disorder ‘randomness ofa system. An example of an increase in entropy isthe dlfusion ofa solute such asa lamp of sugar in a eupof tea. A negative entropy condition would be required to sustain the sugar inthe hot tea asa lump. Similarly, negative entropy is a measure of the “holding {ogether’ of biomolecules in their characteristic shape. Notice that AG ives no information about the rate of a reaction, only if it ean occur spontaneous! and ‘Types of reaction Inan exothermic reaction, heat is given off: Therefore, AH is negative In an endothermic reaction, AH is positive since heat is absorbed by the system from its surroundings. According tothe fist law, no enerey ‘can be lost from the system during the chemi: ‘exergonic reaction, the energy lost during the reaction is conserved as heat reaction, and in an Standard free energy changes in fre Che energy during a reaction are influenced by the pressure temperature and the iil concentrations of the reaetans and prods, Biological reactions are also influenced by pi. In order to sandandie fee eneray changes, standard conditions have been adopted: the temperature is taken to be 25°C (298 K); the pressure is Vat (1, 1325 10" Pa: the inital concentrations of reactants and prodvets is | mol the pH istaken a 7 the molar concentration Ot H.O is taken as unity (1 mol). AG under these conditions i expressed as AGY. “Thee isa formula for AG”, (cL) “[ATIB) o whore AG i the free enersy change for a reaction, Ris the g3s constant, T isthe absolute temperature and fog, isthe natural logarithm, rom Ea AG" = 2.303 RTIog,,K,, 0 AG = AG" + Rlog, (2) and (6), it can be worked out that‘Note: the gas constant (R) is also called the universal molar gas constant, and has a Value of 8.134 J/mel per K.Itmeans tht al gases, have the same kinetic enengy fra ixed numberof gas molecules and at ‘given temperature. The absolute temperature is neasutedin Kelvins (K), Theoretically, the lowest possible temperature is-273 K. IF K.,is less than 1, AG” is postive. This means the reaction will not occur unless energy is applied. The reaction is said to he endergonic. If K_ is greater than I, the reaction will occur spontaneously because AG” is negative. The reaction is said to be ‘exergonic (see above) 630 Chemical reactions II OXIDATION-REDUCTION REACTIONS Ovidation-reduction (redox) reactions are important in biochemistry and the mitochondrial electron transport chain, for example is better understood if the basie principles of redox reactions are known | beforehand. Any substance that donates an electron to another in a cheticl reaction called redectant. Any subiance that accepisan | electron clled an onldan. Consider the equation 2A 42H 204 H, w which cam also be writen as two hal-oquations: 2k-42A 426 ® 2H 29H, a | ‘Compound A has been oxidized, and! H° ions have been reduced t0 ‘gaseous H., Electron flow is part of electrochemical change, and is associated ‘with an electromotive force (emt) that drives the reaction. This force can be measured, an is expressed as the redox potential (and isa ‘measure of the likelihood that a redox reaction will accur. E can be ‘measured for reactions under standard conditions (1 atm of pressure: 298 K (25°C): pH 7) when itis termed E”, and expressed in volts Measurement of E", is measured as an electron flow between two half-ells one of which isthe sample cell, containing a solution of I mol concentration ofthe ‘oxidant tobe tested, linked by abridge toa standard reference half-cel ‘which contains | mol/! solution of Hons in equilibrium with H, gs at I atm. The voltage of the reference solution is taken as O V under these conditions. Eleettons will low from reduetant to oxidant through clectrodes immersed in the solution, and the emf is measured in a voltmeter, ‘Nove: although the potential of a standard H, electrode is set at OV, this is taken for pH = 0, When pH = 7.0, the E”, is 0.42 V, Substance A in Equation (1) has a lower affinity for electrons than does Hund the £', will be negative. A postive E', means tha the substance has a higher affinity for electrons than does H,. A strong. ‘oxidizing agent, such as O,,has.a positive redox potential while song reducing agent, such as nicotinamide adenine dinucleotide (NADH), hhas a negative redox potential “The larger the postive E’, the stronger the oxidant, i. the higher is its alfinity for electrons; in other words, it will tend to be educed. The ‘more negative the E", is the more easily will the reductant release electrons, ‘The tendency ofthe overall redox reaction ta happen can be calculated ply by subtracting the redox potentials ofthe two half-reactions. For example, consider the exercise below. Assume that we dissolve in | Lofisilled H,O,a1 28°C, I-g molecule (1 mol) of each of oxaloacetate, acetate, malate and acetaldehyde Predict which substance willbe oxidized, and which wll be reduced ‘Which wil be the oxidant and which the reductant? oxaloacetate + 2H" + 26-9 malate — where £’,=-0.10V (i) acetate + 2H" + 2e acetaldehyde — where £", “iy Equation i) has the more postive redox potential. Therefore it will proceed more readily asa reduction, eas itis writen, Therefore, Equation (i will proceed in reverse as an oxidation. Solving oxaloacetate + 2H" + 2e 9 malate ‘acetaldehyde — acetate + 2H" + 2e ‘pgaloacetate + acetaldehyde — malate + acetate (Oxaloacette gains electrons and is reduced to mate and acetaldehyde loses electrons and is oxidized to acetate, Therefore, acetaldehyde is the reducing agent and oxaloacet Alo: isthe oxidizing agent 0.10) (0,60) =-0.10+ 0.60 405VFREE ENERGY OF OXIDATION “The free energy of oxidation, AG®, is given by AG" = -nF AE : a Where wis the number of electrons transferred por mole and F is the Faraday constant (96 500./V equiv) Note that A, must be positive in ‘owder for AG” tobe negative THE NERNST EQUATION “The Nerst equation ean be used to calculate the redox potential under non-standard conditions, in which the concentrations of the reactants are present at non-standard concentrations RT... loxidized form) = £, + SLiog, [esidized form) B= Bo + ip Ered form] 8 Expressed in terms of log, pa f+ 22ORT gy, lossized form) wo 8 reduced form) Therefore the Nernst equation can be expressed as o COUPLED REACTIONS Biochemical reactions vey often require energy: they may not ‘20° by themselves, for example the formation of macromolecules such as rueleic acids and proteins from their respective nucleotide and amino acid subunits. This sor of elaboration requires work, which in tum requires energy. The energy is supplied by exergonie reactions. Exergonie reactions do the work that drive the endergonie reactions. “The cell has enzymes that catalyse exergonic reactions: some of ‘the energy produced by the reactions i trapped by coupling the reaction to an endergonic reaction, which generates so-called energy-rich ‘compounds such as ATP,31 Enzymes | Propertis of enzymes-bological catalysts Level of acbaton energy requed for acon Speci or Enzymes are proteins which catalyse chemical reactions, They can ‘operate in the living cell, or in the test tube under the correct ‘conditions, and are even put into washing powders for digesting food stains in clothing. Enzymes inerease the rate at which a reaction reaches equilibrium, although they do not alter the thermodynamic properties of the reaction, for example the equilibruim constant of the reaction, ACTIVATION STATES Enzymes, like other catalysts, are not changed themselves after the reaction although they may be temporarily altered in structure while the reaction is proceeding. Enzymes make reactions possible in the boy, at rates which allow the cells to live. Normally, at 37°C and at ap of 7, most ofthe reactions necessary for life would go toe slowly.During a chemical reaction, the reacting chemicals pass through an energy state higher than that of either the reaetants oF ofthe products The reactants need to attain a certain aetivation energy in order to reach so-called transition state, and the at of the reaction will depend (om the rate at which individual reacting molecules build up activation ‘energy to the transition state, Enzymes, like other catalysts, actually reduce the amount of activation ‘energy required. They achieve this by providing altemative pathways, for the reaction; these pathways require less activation energy than would ‘be needed in the absence of the enzyme. PROPERTIES OF ENZYMES Substrate and reuetion specificity Unlike inorganic catalysts, such as platinum, which can catalyse a whole hhos of reactions in the test tbe, the biological catalyst, the eneymes, ate highly specific. They will recognize one oa few chemically closely related substrates. Substrates may be defined as substances on which enzymes actin biochemical reactions. Similarly, enzymes wil catalyse specific types of ‘Chemical composition Enzymes are macromolecules. They are almost ll proteins (although it has been shown that RNA can eatalyse certain reactions). They are composed of amino acid chains, whose specific sequences determine the folding, shape and function of the enzyme. FACTORS AFFECTING ENZYME ACTIVITY pH ‘The pH cam affect biochemical reactions in a varity of wa 1 extremes of pH may radically alter enzyime structure, and thus denature the enzyme: 2 the pH may affect the degree to which the substrate is ionized and thus affect the rate of the reaction: 3 the pH can affect the binding of the enzyme and substrate; 44 the pH can alter the reactivity of the enzyme during the catalytic process. Only (3) and (4) will be considered here. ‘Most enzymes are active only within a fairly limited pH range, and they have an optimum pH at which their aetvity is greatest. The pH optimum will depend on where in the organism the enzyme is physiologically active. For example, pepsin, a digestive enzyme, ‘operates in the stomach in the presence of hydrochloric acid. andits pit ‘optimum is around 2. Lysosomal enzymes have a pH optima of around '5.the pH in the lysosome. ‘Temperature All chemical reactions are increased asthe temprerature is increased, including enzyme-catalysed reactions. But, when enzymes are heated above 40°C many of them begin to become denatured, which results in ‘fallin activity anda fallin the rate of the reaction. Once the enzyme is denatured, comparatively litle more of the product will be formed, 0 ‘matter how long the reaction is left to proceed. cLass FICATION OF ENZYMES ‘The Commission on Enzyme Nomenclature ofthe International Union ‘of Biochemistry established a classification of enzymes into six clases, 1 Oxidoreductases: these catalyse oxidation-reduction reactions. An example is alcohol dehydrogenase, which oxidizes alcohol to acetaldehyde, 2 Transferases: these catalyse group transfer reactions. The ‘groups transferred include methyl, ketone, nitrogenous or phosphorus ‘groups. Examples of a transferase are hexokinase and methyl transferase. 3 Hydrolases: these catalyse hytirolytic reactions. They cleave O-P, (C-Nand C-O bonds. Examples are the peptide hydrolases, which cleave peptide bonds. 4 Lyases: these catalyse the reversible addition of groups to double ‘bonds or formation of double bonds by removal of groups, For exaruple, they may remove ammonia, CO, or H,O during the reaction. An example is pyruvate decarboxylase, which decarboxylates a keto-acid to yield an aldehyde with release of CO,, § Isomerases: these enzymes catalyse different kinds of isomerization ‘hich involves the rearrangement of a molecule to yield one with differen physical and/or chemical properties. There are diferent types of isomerase. The epimerases and racemases catalyse inversion at asymmetrical carbon atoms. For example, lactate racemase catalyses the conversion of lactate to b-actate, The mnutases catalyse the transfer Of groups within a molecule. For example, phosphoglycerate mutase produces 3-phosphoglycerate from 2-phosphoglycerate. 6 Ligases: these are also termed symtetases, They catalyse the condensation of two molecules, and the reaction is coupled to the cleavage of a high-energy phosphate bond, such as is found in ATP. An example is pyruvate carboxylase, which condenses pyruvate and bicarbonate o yield oxaloacetate, which is coupled tothe conversion of ATP to ADP. 632 Enzymes Il Coenzymes and enzyme action Models of enzyme action Prot fda | i i Coenzymes vulpes Bema emo iden spats | Trareainase Tansanioase Ni Be acl ot proton kinase C stp showing acraon and | sibsatespectty > pseudosubsrate s R= repulse } S-= subsrala poset MECHANISM OF ACTION Protein folding Enzymes are globular proteins consisting of one or more polypeptide chains which fold into a three-dimensional structure, depending on the amino acid sequence. The amino acids forming an active site may be far apart, bu are brought into proximity through chain folding. Active sites om the enzyme surface include the substate-binding, regulatory and catalytic sites, The nature and arrangement ofthe amino acids ata site determine, largely, the functional specificity of the active site. Substrate-enzyme complex formation The substrate is held atts binding site by non-covalent forces, including and hydrophobic bonds, and electrostatic and van der Waals forces. The binding site isso specifi fora substrate that often it will, bind only one isomer ofa diastereomeric pair. Some enzymes are nota speeitic. Glucokinase will bind only glucose, while hexokinase will bind and catalyse the phosphorylation of 2-deoxyglucose, fructose, slucosamine, glucose and mannose, although not all at the same rate. Ieis believed that when the substrate makes contact withthe active site the site changes its conformation to accommodate the substrate ‘Thisisthe induced fi model, Possibly the induced ft mechanism places strains on the substrate, thereby lowering the activation energy required forthe reaction to occur. Mechanism of eatalysis “The substrate-binding ste is usually where some or all ofthe catalytic ‘reaction occurs. Chymotrypsin isa digestive enzyme catalysing the hydrolysis of dietary proteins in the small intestine, cleaving peptide ‘bonds at the carboxy! side of aromatic side chains of phenylalanine, ‘ryptophan and tyrosine. There are two main steps inthe reaction. I The substrate binds to the enzyme at a chemically active site dominated by triad of three amino acids. These are Asp 102, His $7 and Ser 195, which form a eatalyti triad. The substrate and the triad become bound by hydrogen bonding, 2 The susceptible peptide bond of the substrate is cleaved through the action of His $7 and Ser 19S, andthe peptide substrate is hydrolysed to yield an acid and an amine. Regulatory sites on enzymes Enzyme activity may be regulated by sites on the enzyme itself. Examples are enzymes that phosphorylate proteins, the protein kinases. Protein kinase C (PKC) a serinefthreonine kinase, is activated by DAG, Phospholipids and calcium ions, and is believed to mediate cellular ‘events following activation of cells by hormones an second messengers. PKC appears to be a member ofa family of PKC isotypes. ‘The enzyme has functionally distinct regions: a substrate pocket; an amino-terminal regulatory site: a pseudosubsteate site within the regulatory domain: ond a carboxy-terminal catalytie site. The pseudosubstrate site consists of sequence of amino acids resembling the substrate, but without a serine/threonine residue which can be phosphorylated. Instead, it contains an alanine residue. Inthe inactive state the pseudosubstrate ste occupies the substrate pocket within the catalytic domain and inactivates it. Activation ofthe enzyme causes a conformational change and dissociation of the pseudosubstrate from the substrate pocket. But, the pseudosubstrate still interacts with the substrate pocket, allowing only certain substrates to gain aceess wo the Pocket, thus playing a part in determining substrate specificity COFACTORS Cofactors are chemicals that assist or are necessary for enzyme action. Cofuctors become attached to the enzyme, usually at the catalytic site, and may enable the binding of the substrate and/or the catalytic process. There are two main groups: (i) coenzymes; and i) prosthetic groups. Coenzymes CCoonzymes may be metals, e.g. cobalt, copper. iron, Mg™, manganese (Mn) or Zi For example, Mg or Ma are needed forthe reduction ‘of the high negative charges of ATP during the kinase-catalysed phosphorylation reaction. The phosphorylation of glucose to glucose ‘6-phosphate provides an example ofthis type of reaction. Coenzymes may be organic molecules which are derived from vitamins. Mechanism of coenzyme action. Coenzymes, such 3s favine adenine dinucleotide (FAD) may be required as profon and electron acceptors FFAD is reduced to FADH, during dehydrogenation reactions, and in & ‘separate reaction is oxidized back to FAD, which is ready 10 act as coenzyme again, Therefore, FAD is not only a coenzyme but a substrate two, Since the coenzyme is altered during the reaction, itis sometimes refered 10 as a second substrate forthe enzyme. Vitamin B, (pyridoxine) is an cc-amino group acceptor which can transfer the amino group ton c-keto-aci, thus forming anew amino acid, Prosthetic groups Prosthetic groups are non-protein, which bind covalently tothe enzyme at its active site, These include metal ions and organic molecules such a biotin. Biotin is vitamin B,. and is required forthe incorporation of CO, into organic compounds. It does this by acting asa carter for CO, Biotin binds covalently toa lysine residue atthe catalytic ste of the ‘enzyme, and accepts -COO,, usually from HCO, The -COO is then ‘passed rapidly tothe substrate wo form acarboxylated compound. Bictin needs ATP to bind CO.. ‘Metals such as 21° may function as cofactors by binding the substrate, and/or by promoting catalysis, Proteins that contain covalently bound metal fons are termed metalloproteins. MULTISUBSTRATE REACTIONS Enzyme reactions in which one enzyme may be able to bind more than one substrate may he sequential or what is sometimes called ‘ping-pong. In sequential reaction, one substrate may need to be ‘bound before another, for example alcohol dehydrogenase frst binds ethyl alcohol prior to binding the cofactor NADY. The reaction is said to be ordered, whereas one in which two substrates can bind in any ‘order is aid to be random. In a ping-pong reaction, an enzyme binds a substrate, converts it to a product which is released and then binds another substrate. An example is the transaminase reaction, where a Ketovacid is released as product and another keto-acid bound as a substrate, 033 Enzymes Ill Ces Constant substrate concentration Progress curve In wey % Proc med (na) ‘Substrate saturation curve Lineweaver-Burk plot str cect] (nt Maan ey | Fig degraded, or as equilibrium is approached. From the graph obtained, called a progress curve, we can derive the inital velocity, yp, There ENZYME KINETICS the enzyme concentra is a direct relationship between Enzyme-substrate interaction La aan the reaction mixture, in tha the rate of the eaction will double when jon is doubled, for a given concentration af When enzyme and substrate react together, the rate of the reaction the enzyme concentra will increase at first then slow down with time asthe enzyme becomes substrateSubstrate saturation curve {fyi plotted against substrate concentration a a given concen of enzyme, then acurve i obtained whose shape we cal rectangular hyperbole. Initially, the rate ofthe reaction v, is directly proportional to the substrate concentration (S|. (Mathematicians call this “first- forder' kinetics.) But, as [S] increases, the rate eventually reaches a limiting maximum, V,_.. The maximum reflects the fact that all the binding sites on the enzyme are taken up of saturated, and the rate ‘could be inereased only by adding more enzyme. This curve is called the substrate saturation curve. (Mathematicians call the "bend? in the curve “mixed” of first- and zero-order kinetics.) The flat part of the curve reflects the fact that there is no further increase in rate no matter how much substrate is added. (This is called “zero-order” kinetics.) Enzyme units Enzyme activity is measured in units established by The Commission ‘on Enzyme Nomenclature of the International Union of Biochemistry (SI) The units the katal (kat). One katal is the quantity of enzyme in the presence of which I mol of substrate is converted per second. Its ‘more common, however. to express enzyme activity in millikatals or rmierokatals, since L kat is equivalent tothe activity of about 1 kg ofthe pure enzyme. In many textbooks, enzyme activity is expressed as ‘micromoles of substrate that are converted per minute to product under siven assay Conditions. The standard unit of enzyme activity. U. isthe ‘amount of enzyme catalysing the formation of | mol of substrate per minute. The specific activity of an enzyme preparation is the number ‘of enzyme units per milligram of protein. Ths gives an indication of the purty ofthe preparation. ‘The formula of the substrate saturation curve “The substrate saturation curve allows us to derive equilibrium constants that characterize the enzyme-substrate reaction forgiven conditions of pH and temperature. We can arbitrarily define a constant, the K,. ot Michaelis constant, whichis the substrate concentration when the rate half the maximum rate (see below). The equation that describes the curve was derived by Michaclis and Menten, and the equation they derived is called the Michaelis-Menten equation: Vu 81 K+(S1 o fy, isploted agains {5}, rectangular hyperbole is obtained. tis easy to calculate that when v, = V__ 2, thea K;, = [5] In order to obiain values of K., and V,_. the Michaelis-Menten equation can be linearized 1 Invert Equation (1) L_K, +181 6 Vaal] a 2 Separate and simplify to give the Lineweaver-Burk plo: RE wan ® ‘The Lineweaver-Burk plots the equation of asraight line (y = mx + 0) ‘when 1/ (9) is plated against 1/[S] (a) with gradient (m) = KV, and the intercept (c) on the ordinate is 1... UK, can be rea directly {rom the graph where the line crosses the abscissa. Although not presented here, there are other methods of Tinearizng the “Michaelis-Menten equation, which are generally favoured by biochemist34 Enzymes IV Enzyme inhibition Fie MI KINETICS OF ENZYME INHIBITION Enzymes may be inhibited from acting by small ons or molecules which {orm par of regulatory contro systems or by drugs Inhibition of enzyme action may be irreversible or reversible. Reversible inhibition may'be competitive or non-competitive: the nature of reversible inhibition can 1% be determined using enzyme kinetics and the Lineweaver-Burk plot ‘Some small molecules may control enzyme activity by bi allosterie sites, which inhibit or activate the enzyme. Many enzymes are oligomers, being formed of identical subunits or protomers, each with a substrate-binding site. Activation of an allosteric site on one ‘protomer may increase substrate-enzyme aflinity on other protomers ‘a process called co-operativityIrreversible inhibition reversible inhibition is permanent enzyme inhibition, usually due to the covalent attachment ofa chemical tothe enzyme at one or more of its active sites, oF at another site which alters the conformational shape ofthe enzyme. The orpanism has to preuce more enzyme to replace it Examples of irreversible inhibitors are heavy metal ions such as mereury ions. Nerve gases such as the organophosphorus compound di ‘sopropylphosphofluordate (DEP) bind ireversibly tothe specifi serine in the active centce of a large group of hydrolases, for example acetylcholinesterase. Reversible inhibitors Reversible inhibitors bind to enzymes non-covalently and are able to dissociate thus leaving them free to catalyse substrates. Reversible inhibitors can be removed by dialysis. Competitive inhibition ‘A competitive inhibitor competes with the substrate for its binding site om the enzyme, and is often structurally similar to the substrate ‘Once hound, the inhibitor may itself be converted to product, or ‘occupy the site until it dissociates from it. The action of the inhibitor can be overcome by increasing the concentration of the normal Substrate at the binding site. The Lineweaver-Burk plot in the presence ofa fixed concentration ofthe inhibitor reflects competitive inhibition through an apparent increase in the K,, and an unaltered ‘An example of a competitive inhibitor is malonate, which competes, with sucinate ats hiding sve on he enzyme succinate dehydrogenase \where succinate is normally converted to fumarate. Ifthe concentration of suceinate is inereased, it will displace malonate from the enzyme. Examples of metabolized inhibitors are the antibiotic sulphonamides, suet as sulphanilamide, which bind to dihydroprerate synthetase, a bacterial enzyine that synthesizes folic acid from p-aminobenroate, Which is necessary for bacterial growth. The enzyme converts the sulphonamide toa compound that cannot be metabolized to folic aid, and the bacteria die, Non-competitive inhibition Non-competitive inhibitors usually bind to the enzyme a sites other than that which binds the substrate, and the substrate does not compete with the inhibitor. Therefore, although the substrate may sill be able 0 cecupy its own binding ste on the enzyme, its not converted to product. The inibitor-enzyme-substrate complex is sometimes called a ‘dead- end" complex, since it is catalytically inactive, Non-competitive inhibitors in effect remove enzyme from the available pol, and the Lineweaver-Burk plot reflects this in thatthe Kis unchanged but the Vs reduced, ALLOSTERISM Allsterism is a word used to describe enzymes that have binding sites for molecules, usually of low molecular weight, other than those where substrates hind and are converted to prxuct. These small molecules ar termed ligands, A ligand isa molecule tha binds toa binding site on & ‘macromolecule, such as an enzyme orareceptor These allosteric binding sites are very offen where enzyme activity is controlled. Ligands dha ‘rind o allosteric binding sites may be allostere activators or allosteric Inhibitors. The product ofan enzyme may itself bean allosteric inhibitor, binding to and inhibiting an enzyme funher hack the chain of metabolic pathways that produced i. This isan example ofa physiological control ‘mechanism whereby metabolic processes are regulated, Classes of ullosterie enzymes Allosteric enzymes have been classified as K elass and V class, ‘depending on how their allosteric Tigands affect the kinetic constants Inthe presence oftheir allosteric ligands, K-class enzymes yield plots showing an altered K, and unchanged ¥,., (asi seen with ‘competitive inhibitors of enzyme action). In other words, an allosteric inhibitor binds tothe allosteric site, and the enzyine reacts by losin affinity forthe substrate In the presence of their allosteric inhibitors, V-clas enzymes yield plots showing an unchanged K,, and lower V,,. (as is seen with non ‘competitive enzyme inhibitors). ‘Mechanism of allosterism “The mechanism of allostersm is not well understood, but it is believed that when ligand binds to an allosteric site it alters the conformation of the enzyme so that the anit ofthe substrate (or some other ligand) for itsbinding site onthe enzyme is either increased or decreased, depending, ‘on whether the ligand isan allosteric activator or inhibitor, respectively Ia ligand increases the affinity of another protomer for the same ligand, this termed s homotrophie interaction. I'he igand increases the affinity of another protomer fora different ligand, this is termed & heterotrophic interaction Co-operativity is the term used to deseribe the effeet that one protomer ean exert on another, as happens when a ligand binds. Co-operatvity reveals ise in the substrate saturation curve, which is sigmoidal, and the formula of the line is given by the HIM equation, ‘which can be linearized. Allosteric activators will shift the curve tothe Jot, while inbibitors will shift the eurve to the right, The binding of O, ‘olyprovides another example of postive co-operativily anda sigmoidal curve. Note, however, that sigmoidal curves will always be obtained ‘with mullisubunit enzymes, even i the absence of eo-operatvity Allosteric effects ean occur on one protomer without 3 co-operative effect on other protomers. For example alcohol dehydrogenase isa zine- containing metallopeoiein which reduces acetakdebyde to alcohol, and consists of protomers which are independently activated allostericallyFig 381 35 Digestion: basic principles and cell types INTRODUCTION When food is ingested, its not in a form that ean be readily absorbed by the cells ofthe alimentary tract. For efficient absomption to oecur, the polymeric foodstuffs are hydrolysed into their constitutive residues, ‘which ean be taken up more easily ino the epithelial cell Digestive enzymes are released in controlled secretions from specialized organs and cells of the alimentary tact. These secretions also contain composi ‘enzymes, Daily, 1-21 of saliva are secreted into the mouth, 2.51 of ons of solutes to maximize the activity of the {gastric juice are secreted ito the stomach, 1-31 are secreted into the small intestine by the epithelial cells of the intestinal wall and || of bile and I of pancreatic juice drain directly into the duodenum, Due to the differences in the composition of the seeretions, the pH of the tract varies widely. Int the pH is usually 4.0 due (othe secretion of hydrochloric acid, but in the duodenum the pH rises to 6.5-6.8 with the addition of bicarbonate ions in the pancreatic juice. The epithetial cells of the intestinal wall, therefore, are not only ‘adapted tothe absorption of digested foods but also (othe maintenance‘of a luminal environment in which digestion and absorption can occur ‘at maximal rates Solute transport ‘There are two distinct pathways for solute transport () the solutes may pass through the tight junctions which connect the epithelial cells of the intestinal wall to cach other (paracellular route); oF (i) they may pss directly through the epithelial ells themselves, so passing through the luminal membrane and the basolateral membrane (transeelfular route), ‘The luminal membrane contains many protein transport molecules that are specialized for uptake or secretion of solutes and of digestive enzymes. The basolateral membrane is more typical of the plasma ‘membrane of most cells, while also showing transport systems for the i absorbed into the cell from the lumen. driving fore for almost al of the transcellular solute inst an electrochemical gradients the Na‘/ K'~ATPase in the basolateral membrane. The actions of this energy-consuming. ‘ranspor protein are responsible for low Na* and high K* concentrations in the cytosol and an electrical potential ofthe cytoplasm of ~60 mV ‘compared with the extracellular fluid. The potential difference is caused by the asymmetrical transport ofthe ions by the protein (three Na ions are transported out, while two Kions ae transported in). exit of nutri “The domina MECHANISMS FOR THE ABSORBTION OF SOLUTES. ‘The Na* in the lumen ofthe small and large intestines orginates from both the dietary intake and the secretions of the exocrine glands whieh drain imo the intestinal tract. The uptake of Na* into the enterocytes along with CI is therefore crucial to the maintenance of overall electrolyte balance in the body. The transport of Na” i also intimately involved in the uptake of digested food molecules such as glucose and amino acids (see Fig. 35.1), ‘The ight junctions connecting the epithelial ces in the large intestine are much less permeable to Na* and HO than are those in the small, {ntestine. This correlates with the Na*-scavenging function ofthe large intestine and the bility of the small imtestne to secrete Ne Uptake of solutes ‘The luminal transporter involves the uptake of solutes such as glucose or amino acids: Na* flows into the cell down the clectrochemical gradient, established by the Na‘/K°-ATPase, through, « cotransport protein which carries the solute across the luminal membrane against its concentration gradient. This cotransporter can transport both ways, bt is influenced in one direction by the negative ‘potential in the eel In the small intestine, six specific carrer-mediated cotransporter ‘systems for t-amino aids have been identified: () acidic amino acid, ‘eg. aspartate; (i) basic amino acids, eg arginine: (i) uncharged amino acids with polar or shor side chains, eg threonine i) uncharged amino acids with hydrophobic oF aromatic side chains, ¢,g. methionine: (©) imino acids, e.g. proline: and (vi) amino acids, ee. taurine, Deftuctose, glucose and o-galactose are the main monosaccharides resulting from digestion of carbohydrates. The latter two are absorbed by a monosaccharide cotransporter on the luminal membrane as described above, while -fructose is absorbed through the huminal membrane by facilitated difusion mechanism which is Na* independent. ‘The basolateral transporter isa different type of transport system “which facilitates the passage ofthe absorbed solute from the epithelial cell into the bloodstream. The basolateral transporter is not a cotransporter. MECHANISMS FOR THE SECRETION OF SOLUTES “The secretion of solutes other than the digestive enzymes themselves is Important for a number of reasons. 1 The changes in osmotic pressure that result from the movement of the solutes from the epithelial cell layer eause the movement of HO imo the lumen of the tract. The HO is necessary to provide a more effective medium for digestion. 2 Enzymes in different areas of the digestive tract require ditfering conditions for their maximal efficiency, and the controlled secretion of acidic and basic ions can provide the enzymes with the environment tha they need. [Nar and CI- can be secreted by most of the epithelial cells of the intestinal tract, but the pH of lumen is controlled by specialized cell types that secrete bicarbonate ions into the pancreatic juice and hydrochloric acid into the stomach, PATHOPHYSIOLOGY Vibrio cholerae isan infection of the gastrointestinal tract. Toxins that, are produced by the bacteria cause excessive secretion of electrolytes by the stimulation of a cAMP regulatory pathway. The resulting diarrhoea can be life-threatening due to loss of essential electrolytes and H,O. Treatment is based on the fact that the Na’=glucose ‘cotransporters not regulated by 2 cAMP pathsay and so administration ‘of oral glucose will cause absorption of Nav n36 Digestion of protein and carbohydrates Digestion of proteins and carbohydrates Pancreas | @ —— Panaeaic $ “Dress evi this, 10-20 g of protein are secreted as enzymes and about 20 g of protein as mucosal cells that ae sloughed off from the intestinal surface into the 1 70-kg man eats 80-100 g of proven daily. In addition to lumen ofthe intestine. Vall al ofthis protein is digested and absorbed, DIGESTION OF PROTEINS On ave %“The first stage of protein digestion occurs in the mouth with the ‘mechanical breakdown of the food by the teeth. Ths provides a larger surface area for the later stages. Protein digestion in the stomach In the stomach, hydrochloric acid secreted by the parietal cells kill invading bacteria and causes unravelling of the protein chains or denaturation, providing an increased surface arca for digestion. The first stage of enzymatic digestion also occurs in the stomach. When digestive enzymes are released into the lumen, they are in an inactive form czymogens) so that they will not damage the mucosal surfaces of ‘the epithelial cells that line the intestine. Pepsinogen is released by _zymogen-releasing cells inthe stomach, and is the precursor for the digestive enzyme pepsin, Cleavage of the peptide bond between residues “44 and 45 of pepsinogen to release pepsin can occur spontaneously at 2 PH more acidic than 5 (as provided by the hydrochloric acid (autor activation)), or by active cleavage ofthe peptide bond by pepsin itself (autocatalysis). Pepsin is stable only in the stomach and cleaves peptide bonds onthe -NH, acids, €. tyrosine (Tye), phenylalanine (Phe). The large peptide fragments and amino acids which result stimulate secretion of digestive enzymes into the small intestine, Digestion of proteins in the small intestine Digestion of proteins in the small intestine i triggered by the controlled release of enterokinase from duodenal epithelial cells, and is ‘dependent on the release of bicarbonate ions, which neutralize the acid from the stomach, Enterokinase cleaves a hexapeptide from trypsinogen, one of the zymogens released from the pancreas, to form trypsin. Trypsin, in addition to its own autocatalytic powers, cleaves Peptide fragments off other pancreatic zymogens to activate them. ‘The activated pancreatic enzymes hydrolyse peptide bonds a different sites along the polypeptide chains. There are two earboxypeptidases that release amtino acids from the carboxyl terminal of the protein. ‘Trypsin, chymotrypsin and elastase are endopeptidases and will digest, the protein from within the chain. Oligopeptides resulting from the action of the pancreatic enzymes are further digested by endopeptidases, aminopeptidases and NAD(P)H + He FAD +2H'+2e > FADE, [NAD * and NADP’ are abe 1o move freely between different dehy dro- genase enzymes, but FAD is aached covalently to succinate dehydro genase. As the coenzymes are reduced, they are effectively absorbing the potential energy which was contained in the food molecules, because the reoxidation of the eoenzymes NADH and FADH, fuels the synthesis of ATP. “The reduced coonzymes transfer their electrons into the electron transfer chain (see Fig. 38.1). This is a series of proteins on the inner mitochondrial membrane organized in onder of progressively jeteasing redox potential, NADH ean reduce (and so transfer its ‘lectons to) the frst complex of the chain, while FADH, ean only feduce the second complex of the chain, The driving force generated by the increasing redox pocentials of adjacent components of the chain carries electrons through the chain until, finally. complex IV somehow accumulates four e and reduces O,, which then combines with H’ to form H,0. AS the electons mave down the redox potential aradient, energy is released and at three sites along the chain (complexes I II and IY) the energy released is sufficient to pump H° across the inner mitochondrial membrane from the matrix to the intermembrancous space. The outer sueface of the inner membrane subsequently becomes 140 mV more positive than the inner surface ‘of the membrane and the intermembrancous space becomes 1.4 pH units lower than the mateix. As a result, @ proton motive force of approximately 200 mV develops across the inner mitochondrial mernbrane, This membrane is, however, mmpervious wo HY and the only ‘way forthe Hr to ross is via proton channel (F,) which is coupled {to ATP synthase (F,). The ATP synthase enzyme is activated by the passage of H” and eatalyses the phosphorylation of ADP to ATP with the passage of three protons through the channel. This method forthe generation of ATP from ADP and P, is known as oxidative phosphorylation. Getting ATP out of the mitochondria and ADP io the mitochondria costs one H*, and so ittakes four H’ to produce ‘one ATP outside the mitochondria. FADH, can only reduce complex so its reoxidation yields only two molecules of ATP while that of NADH yields about three. Complex I is composed of » NADH dehydrogenase with a flavin mononucleotide cofactor and various Fe-S proteins. Complex Tis composed of various FADH dehydrogenases and Fe-S proteins, Coenzyme Q (ubiquinone) shuttles from I and II Il. Complex TI is composed of cytochromes b and e,. Cytochrome e shuttles © from complexes II to TV. Complex IV is composed of eytochromes anda,39 Glycolysis and gluconeogenesis Glycolysis and related reactions Ghose i cose & phosphatase Guomse ghosts { Prosphogbense ‘somecase Fructose phosphate Fras 16: Dhysroxacetire ¢ Sp ycorlty se ‘phosphate rl UDP glucose ce Gyeraeyde phosphate Denyeregerase NADH ne [rmeeoree oe = jmp Peel ee ae 2.3 biphosphoghycerate ‘on S-phosphoglycerate SS m phosohatase | if er gar as suske i : - racer pee iP Pyruvate carboxylase py euat he sae Fig 4 noe NADH Control of phosphofructokinase| ‘and fructose 1,5-biphosphatase [— Presphoucoknase ‘Acer CohGLYCOLYSIS. Giycolysis, which oecurs in the cytosol of the cell, eam be divided into to parts. 1 In the first part, the sicarbon monosaccharide is phosphorylated lwvice and glucopyranose is converted into fructofuranose. The addition of the phosphate groups uses two molecules of ATP. This phosphorylation prevents the sugar from leaving the cell and activates the molecule fr later oxidative energy-producing reactions 2 The initial reaction of the second part of glycolysis splits the six-carbon sugar into two triose phosphate sugars. At equilibrium, 969% of these are in the form of dihydroxyacetone phosphate ‘This is quickly converted into its isomer, elyceraldehyde-3-phosphate, whieh is oxidized to 1.3-biphosphoglycerate. As a result the second half of the pathway is “repeated once for each molecule of glucose metabolized Substrate level phosphorylation, ‘There art two reactions in glycolysis when ADP is phosphorylated to ATP: (the conversion of 13-bisphosphoglycerate wo phosphoglycerate: and (i) the conversion of phosphoenolpyruvate to pyruvate The two reactions shown above phosphorylate ADP by substrate level phosphorylation, which is the formation of ATP by phosphate group transfer from a substrate. The first of these molecules, 1,3 biphosphoglycerate, is formed by the coupling of an endergonic reaction (the phosphorylation of a carboxylate) to an exergonic reaction (the oxidation of an aldchyde —in this ease glyceraldehyde-3-phosphate) The second of the “high-energy” molecules, phosphoenolpyruvate {generates sufficient fee energy to phosphorylate ADP by its conversion oan intermediate, enolpyruvate, which can then form pyruvate. The second half of glycolysis will therefore produce four ATP, while the first half metabolizes two ATP. 1 summary slucose + 2Pi + 2ADP + 2NAD*— 2pyruvate + 21,0 + 2ATP 4 2NADH + 2H" “There area couple of points that should be noted about glycolysis. 1 All the reactions of glycolysis discussed above are reversible apart from three: those catalysed by hexokinase, by phosphofructokinase and by pyruvate kinase 2 No O, is necessary forthe glycolysis to function. This means that in tissues where ©, is low (eg. active muscles), poorly supplied (eg. ‘comes oF where oxidative metaboliso cannot aceur (e.g. erythrocytes hhave no mitochondria), ATP can stl he produced from energy sources In anaerobie respiration, ie. without O,, pyruvate is reduced to lactate This reaction oxidizes NADH, and the coenzyme can then return to the reaction catalysed by glyceraléchyde-3-phosphate dehydrogenase where itis reduced, Integration of galactose and fructose {into glycolysis Galactose and fructose are also final products of the digestion of carbohydrates and ae integrated into glycolysis (see Fig. 39.1, Boxes 1 and 3). Fructose can also be converted to fructose-6-phosphate by hexokinase, but the enzyme's alfinity for fractose is much lower than for ghucose GLUCONEOGENESIS ‘Under homeostatic conditions, enough carbohydrate is ingested inthe ietto supply organs such asthe brain, and ells such as the erythrocyte whieh require glucose as their primary fuel. Under conditions of dcient dietary intake, glucose can be formed from non-carbobydrate precursor, ‘This pathway is called gluconeogenesis and runs in the opposite irection to glycolysis (ue. from pyruvate to glucose). Gluconeogenesis occurs mainly in the liver, but also to a lesser ‘extent inthe kidney cortex. Some amino acids can enter this pathway’ through ther conversion tether oxaloacetate or pyruvate, depending ‘on their intial stracture. OF the products ofthe digestion of tiglyee- rides, only glycerol can enter gluconeogenesis through its conversion to dihydroxyacetone phosphate and subsequently to glucose, Lactate is also used in gluconeogenesis through its conversion to pyruvate Gluconeogenesis pathwa) Although glucose is formed from pyruvate, gluconeogenesis is not Simply a reversal of the reactions of glycolysis, Three ofthe reactions of glycolysis are irreversible and have to be bypassed o catalysed by different enzymes. They ae the reactions catalysed by: () hexokinase; Gi phosphofrueto-kinase; and (i) pyruvate kinase ‘The overall change in free enerey forthe gluconeogenic pathway is positive and four ATP and two GTP are metabolized to fe! the pathway. The reaction catalysed by pyruvate carboxylase occurs in the mitochoniral matrix. The other reactions in the gluconeogenic pathway ‘occur in the eytosol, Oxaloacetate has no carrier through which it can leave the mitochondria and has to be reduced to malate by a NADH- linked dehydrogenase, Malate does have a specie earir and leaves the mitochondria forthe eytosol where its oxidized 1 oxaloacetate which ‘can retum to the gluconeogenic pathway. To convert two molecules of ‘Pyruvate to one molecule of glucose, four ATP and two GTP are noeded REGULATORY ENZYMES OF GLYCOLYSIS AND GLUCONEOGENES Glycolysis is one ofthe central pathways of metabolism as it provides substrates for both energy production and biosyntheses. ts therefore ‘ital that both glycolysis and gluconeogenesis are controlled to prevent wasting of fuel molecules or excessive production of macromolecule precursors, ‘The main step that controls glycolysis and gluconcogenesis is shown in Fig. 39.1, ATP acts at regulatory sites via an allosteric mechanism, thereby preventing excessive energy production when the ATP/AMP ratio is high, Citrate enchanees the action of ATP, thus preventing the ‘ver-formation of earbon skeletons for biosyntheses, whose levels are reflected by citrate levels, Fructose-2,6-bisphosphate acts to stimulate elycolysis when levels of fructose-6-phosphate tse “There are two other enzymes tha playa smaller par in the control of lyeolysis hexokinase and pyruvate kinase. Hexokinase is inhibited by slucose-6-phosphate, but when glucese levels rise over a critical level in the liver, the same reaction can he catalysed by glucokinase, which hasa higher K, for glucose than hexokinase. The product of the reaction catalysed by pyruvate Kinase is pyruvate, which ean be used 10 build molecules or praduce energy by its conversion to aceiyl CoA. Pyruvate ‘Kinase exist inthe forms: L (mostly inthe fiver) M (in muscle) and A (in other tissues). The L form is allosterically inhibited by ATP, thus slowing the production of energy when levels of ATP are high. 840 The citric acid cycle and mitochondrial carriers Mitochondrion dyceol3 phosphate eycogenase COU Ren) os acontte — FAD + FAH Shyarnyanone NADY NADH He torte coast| JP cans MO csoimase rom — ol ne ile i hyegease en seowrae¢ {on eee pant eeKelogvarate | Stren rorraase |X yap “ ‘Malate cand 005 crate nae | Se icin Cok NAD Succinate CoA oor vee Sines GIP. Miso oe Noe —> Fone —> [I ne nono Se oe Fr aaa! oor ate or or ap ATP Syrtease : ‘ or fir i a (| lene «ar GA: Guanes car 5 t : Promiocater ae cor ie we xe | Toews — re Purine nucleotide cyte a INTRODUCTION Products and stoichiometry The ce mci cyte is assis of reactions inthe curs of te Thre are mnie of mp points tht nnd be noted sbot he inectoodta that complete the oxidation ocarcydrtes, uty ais products andsskchlomety of the cic acd eel Gol arid deen ed no daingrcasglacron aries, 1 Wii ont CIE Uae fr dehydrogenation reacloe; wich tal hey ca fuel the ex cai, Pyruvcin the cyovlsshutled csi the edoron of tice NAD” and one FAD. These hydrogen in the cats of to moconda wae Is corre into acel\ carr eit onl ied quetests ho each odor Coa. Acts CoA then emer the ic aid eel by © te reouldlzed inthe cleron wanspor chain forthe cre ley 4 series of modifications to the carbon skele 3NAD"+ FAD + acetyl CoA + GDP. -m citrate, The subsequent reactions mt, which results in the a bic condit O+Pi-> eyo 3NADH + 2CO, + FADH, + CoA + GTP + 2H different carbon atoms leave the cycle in the ylation reactions are those which are c and by isocitrate dehydrogenase, continue. Consequently, the etic acid eycle can only operate under The overall esction achieved by one round 2. TWwo carbon atoms inthe form of acetyl CoA enter the eycle and tw form af CO, The de alysed by a ketogiuara‘The production of 38 molecules of ATP from the complex oxidation of 1 molecule of glucose 3 The two reactions catalysed by citrate synthase and by fumarase each ‘consume one molecule of HO. 4 A molecule of GTP is formed from a molecule of GDP and Pi inthe reaction catalysed by succinyl CoA synthetse. ENZYMES OF THE CITRIC ACID CYCLE AND THEIR CONTROL ‘The citric acid eyeleis vital in he process of energy generation, but i also intricately involved in other fundamental metabolic processes such 45 gluconeogenesis, amino acid metabolism and lipogenesis. Ici therefore important that the eyele be precisely controlled so as not to provide an excess of biosynthetic precursors nor to utilize more fuel than i absolutely necessary. There are four main enzymes by which the eycle is controlled: (i) the pyruvate dehydrogenase complex: (i) citrate synthase: (i) soeitrate dehydrogenase: and (iv) d-ketoglutarate dehydrogenase 1 The irreversible conversion of pyruvate to acetyl CoA by the pyruvate {dehydrogenase complex isnot part of the eittc acid eyele buts essential for its efficient functioning. The complex is made up of three enzymes: (a) E, — pyruvate dehydrogenase oxidatively decarboxylates pyruvate: (by E, — dinydrotipoy! tansacetylae transfers the acetyl residue toacetyl CoA: and (c) E,—diydeoipoy! dehydrogenase oxidatively regenerates 3 Tipoamide. 2 Citrate synthase controls the entry of acetyl CoA into the eyele, and is inhibited allostericaly by ATP 3 lsovitrate dehydrogenase catalyses the conversion of isocitrate to ‘-ketoglutarate. The enzyme is allosterically stimulated by ADP and is ‘competitively inhibited by NADH which can displace NAD‘ 4 «eKetoglutarate dehydrogenase catalyses the conversion of a= ketoglutarate to succinyl CoA. It exists as a three-enzyme complex Similar to pyruvate dehydrogenase, and shares many of is properties, ive, its activity is regulated by its products and also by levels of phosphorylated nucleotides in the cel, THE GLYCEROL-3-PHOSPHATE SHUNT AND THE MALATE/ASPARTATE SHUNT “Te glycolytic rention that oxidizes slyceraldehyde-3-phosphate to 1-biphosphoulycerate results in the reduction of NAD" 19 NADH (Quantities ofthis hydrogen care are limited in the eytsol a thereore [NADH needs tobe reoxidized for glycolysis to be maintained. “The oxidation of NADH in the electron chain occurs in the mitochon- dria, butthe carrie canno pass across the inner mitochon membrane “Thus, two systems have developed so thatthe eytosolie NADHT can be reoxidized inthe mitochondria 1 In the glycerol-3-phosphate shunt, cytosolic dehydrogenase oxidizes NADH as diycroxyacetone phosphate is reduced to glycerol Ssphosphate A mitochondrial dehydrogenase operates in the reverse rection, but reduces FAD instead of NAD 2 In the malate/aspartate shunt NADH is oxidized by the conversion of oxaloacetate to malate. Malate crosses the inner mitochondrial membrane where itis oxidized wo oxaloacetate. Oxaloacetate is then transaminated wo fem aspartate, which moves back into the eyo PURINE NUCLEOTIDE CYCLE ‘The purine nucleotide cycle is used to supply fumarate to the citric aid jeyele when the availability of acetyl CoA exceeds the availabilty of oxaloacetate This imbalance commonly oecurs in skeletal muscle during exercise, and the nucleotide eyele is essential for maintaining {he efficient functioning ofthe citric weid eye 8841 Glycogen metabolism CN ee Glycogen | | Glycogen (Glucose 6 phosphatase synthesis | | breakdown Z Giucnse 6 phosphate on rane Presprapherorutace om re Glucose 1 phosphate Box! boc Simuatont ‘Senator Goonies Gooneness ‘Guzagoner bx2 — sins | E a ne me a the cell as granules, These granules range from 10 to 40/nm in size and eNERODUEIIOM contain the enzymes that are involved in glycogen metabolism. The nin the body are the liver (100 after 2 meal) and skeletal muscle (up to 300 g, although the the intestines, and brain in which glucose is stored. It is com ds (see C xd by a1 4-plycosiic b 36) wit c-,6-linked branch points. Glycogen exis amounts inGLYCOGENOLYSIS 1 Phosphorylase catalyses the removal of a glucose residue by the phosphorylysis of an a-14-glycosidic bond. Phosphorylase requires the presence of the coenzyme pyridoxal phosphate, which wets as an acid-base catalyst, 10 work, 2 Phosphorylase can only break «-1,t-glyeosidie bonds up 10 four residues away from a branch point. The three residues leading to the branch are then transferred to another part of the glycogen molecule by transferase, 3 The a1 G-alycosidic bond forming the branch point is cleaved by 1 6 glucosidase 4 Phosphoglucomutase converts glucose-I-phosphate into glucose- 6 phosphate, viaa glucose-.6-hiphosphate intermediate, which can then enter glycolysis, Inthe liver, intestines and kidney, glucose-6-phosphatase cean remove & phosphate group from glucose-6-phosphate to form slucose, which can then pass out ofthe cell into the bloodstream. In brain and skeletal muscle tissue, glucose-6-phosphatase is not present and glucose-6-phosphate cannot pass out ofthe cells. GLYCOGEN SYNTHESIS ‘The synthesis of glycogen (glycogenesis) follows a diferent pathway from that of glycogenolysis. 1 To headded onto.achain in a glycogen molecule, the glucose residue -must be inthe ‘activated’ form of uridine diphosphate (UDP)-glucose _lucose-|-phosphate + UTP UDP-glucose + pyrophosphate ‘This is catalysed by UDP-glucose pyrophosphorylase, The reaction is driven towards the formation of UDP-glucose by the hydrolysis of pyrophosphate to orthophosphate by the enzyme pyrophosphatase 2 Glycogen synthase catalyses the addition of glucose from UDP- _lucose tothe non-tedueing end ofa chain. The a- 1 4-glycosidic bond is made with the hydroxy! group on C, and UDP is released. Glycogen symthase can only add UDP-glucose to a chain of five or more residues. (Chains smaller than five residues reattached to proteins and are called primers. 3 When a chain contains more than 11 residues, a branching enzyme ‘transfers block of seven toan interior ste within the glycogen molecule, to form a new braneh. The branch point must be at least four resides avsay from any pre-existing branch points CONTROL O} ELYCOGEN METABOLISM “The synthesis and breakdown of glycogen is one of the major controlling Factors that determine the aailability of glucose for energy production inthe body. When there isan excess of carbohydrates in the body, for ‘example afters meal, iis important that glucose can be stored for later use. In periods such as these, insulin s secreted by the endocrine glands ‘of the pancreas and causes glycogenesis, (On the other hand, if a supply of glucose is needed wh really available from the diet, glycogen can be broken down to give ch is not slucose-6-phosphate, which can be integrated directly into glycolysis or glucose, which can pass into the circulation and be taken up by ‘organs that need it, When concentrations of glucose are low in the blood, glucagon is secreted from the endocrine cells ofthe pancreas, land in periods of stressor excitement adrenaline is secreted from the adrenal glands, Both glucagon and adrenaline stimulate plycogenolysis (Note: there are no glucagon receptors in muscle tissue.) Both glucagon and adrenaline work via a easeade mechanism ‘whereby the intial signal by the hormone is amplified manyfold while being relayed to the enzymes of glycogen metabolism. The hormonal signals are dually effective because reversible phosphorylation is used tocontrol the activity ofthe enzymes. So, for example, when glucose is ‘needed, the enzymes catalysing elycogenolysis are phosphorylated and activated while the enzymes catalysing plycogenesis are phosphorylated and inactivated, Control of enzyme activity by phosphorylation ‘The enzyme phosphorylase in skeletal muscle can exist in two forms depending on the phosphorylation of a specific serine residue: phos- pphorylase a and phosphorylase b. Phosphorylase ais phosphorylated ands usually always active while phosphorylase bis dephosphorylated ancl usually inactive. The two forms ae interconvertible by te actions ‘of phosphorylase kinase and phosphatase 1, which also control the ‘activity of glycogen synthase. In ten, the activity of phosphorylase kinase and phosphatase I are also controled by reversible phosphoryla- tion, The activity of phosphatase I is modulated by a protein called inhibitor 1, When inhibitor 1 is phosphorylated (e, by the action of ‘AMP-activated protein kinase it inhibits the activity of phosphatase 1. However, when levels of ¢AMP fll, or when insulin acts onthe cell Inhibitor 1 becomes dephosphorylated and the phosphatase 1 activity resumes, When glucose levels are high, the activity of phosphorylase falls before the activity of synthase rises. This is explained below 1 Glucose enters the cell and binds tothe phosphorylase component of ‘a phosphorylase a phosphatase 1 complex. 2 Phosphatase 1 dephosphorylates phosphorylase a to yield inactive Phosphorylase b, '3 Phosphorylase b dissociates from phosphatase 1 4 Phosphatase I then dephosphorylates glycogen synthase bto convert Into the active glycogen synthase a form, PATHOPHYSIOLOGY Von Gierke’s disease isa deficiency of the glucose-6-phosphatase ‘enzyme in liver and kidney tissues. The resulting inereased levels of slycogen in the affected organs causes enlargement ofthe organs and hypoglycaemia. Cori’s disease is a deficiency of the debranching ‘enzyme in liver and muscle tissues, Incteased levels of glycogen with short branches result and cause similar symptoms to type 1 disease MeArdle’s disease is a deficiency in masele phosphorylase. Only ‘moderately increased levels of glycogen result in the muscle tissue, but severe cramps develop with exercise due to the lack of glucose-6- phosphate available for oxidation, as42 Lipid metabolism | Fatty acid synthesis and oxidation 1005 eM otests Oxidation =c0-8-Cod coo cH ps ‘env Cokes C05 tek yoo! pan ransieae wy Camtine Moccia Cp LtaeeANC?. city-CO-8 canine ¢~ Teansiwaso tl > dey ca al (C00-CH,-CO-S oa (lty- (CHa CHa CHe~GH-CO-S - CoA Fan y= (Crp CH-C~CHy~CO-Sc0” RD tate of popes Hy~ CH ly CO-S Buty AGP ‘When a Cg 20 ACP has ben omed the action of Tioestrase eaves Patate tom ACP (iy (OHehne~ 000+ AGPBREAKDOWN OF LIPIDS Formation of fatty acids and glycerol {rom triglycerides ‘The breakdown of lipids hegins withthe hydrolysis of a cytosolic trigly- ‘erie into one molecule of glyeerol and tree fatty acid. This reaction is catalysed by the enzyme hormone-sensitive lipase, which is activated by glucagon, adrenaline, noradrenaline snd adrenocorticotrophin (ACTH), These hormones and neurtransmiters act viaa cAMP cascade smcchanism which leads to the phosphorylation ofthe enzyme. Insulin action results in the dephosphorylation and inactivation ofthe enzyme. Glycerol can enter glycolysis by phosphorylation to glyceral-3- phosphate. This is then oxidized to form dihydroxyacetone phosphate Fatty acid oxidation ‘The fatty acids are ‘activated’ by the formation of a thioester bond between the carboxyl group ofthe acid to the sulphydryl group of CoA, ‘The reaction is catalysed by acyl CoA synthetase and is driven forward by the hydrolysis of pyrophosphate. The subsequent shuttling of the fatty acid into the mitochondrial matsix and its breakdown (called ‘8-oxidation) are shown in Fig. 42.1. Camitine deficiency or a defect in the translocase enzyme impairs energy production from fat oxidation and results in muscle cramps developing with exercise. The overall ‘reaction forthe oxidation ofa typical Fay acd i shown below: palmitoyl CoA + 7NAD' + 7FAD +7H,0 +7CoA > acetyl CoA + 7NADH + 7H* + EADHL, ‘The complete oxidation of this fatty acid will thus yield 129 molecules of ATP. ‘Many fatty acids occur that contain one or more double bonds inthe hydrocarbon tail and are sail 1o be unsaturated. The enzymes of the S-oxidation chain are stereospecific and the unsaturated fatty acids have ‘be modified before they can be fully oxidized. The modification of «an acid with only one double bond i shown in Fig. 42.1, Box 1, while those of polyunsaturates are shown in Fig, 42.1, Box 2. ‘The rate of breakdown is determined by the availability of substrates, the inhibition of caritine transferase | by malonyl CoA and the Inhibition of hydroxyscyl CoA dehydrogenase by NADH, Ketone body formation When the breakdown of fats exoeeds the breakdown of carbohydrates, the supply of acetyl CoA from B-oxidation exceeds the rate that ‘oxaloacetate can be formed. The acetyl CoA then follows a different pathway; that of ketone body formation Fig. 42.1, Box 3) The ketone bodies, acetoacetate and hydroxybutyrte, diffuse out of the cell and ate common fuel molecules in the bod, especially in cardiae muscle and the renal cortex where acetyl CoA can be regenerated. The regeneration of acetyl CoA from acetoacetate is catalysed by two enzymes: (3) CoA transferase adds a CoA group; and (i) thiolase cleaves acetoacetyl CoA. incorporating another CoA, to form two molecules of| acetyl CoA, THE SYNTHESIS OF FATTY ACIDS ‘There are three major differences between fatty acid synthesis and breakdown: 1 synthesis occurs in the eytosol whereas breakdown occurs in the mitochondria 2 the enzymes catalysing the breakdown are separate whereas those catalysing the synthesis ae joined to form a single polypeptide chain called fatty acid synthase: 3 the reductant used in the syathesis is NADPH whereas the electron ‘donors in the breakdown are FAD and NAD' “The principal reactions of fatty acid synthesis are shown in Fig. 42.1 Essentially, the pathway consists of the repetitive addition of a wo- carbon unit to the forming fatty acid by the condensation of a three carbon unit (malonyl CoA) tothe end ofthe fatty acid tail and the release of CO, “The committed step ofthe pathway isthe ireversible conversion of acetyl CoA to malony! CoA. The enzyme catalysing this reaction, acetyl CoA carboxylase, has @ prosthetic biotin group attached, which is initially carboxylated by a biotin carboxylase subunit ofthe enzyme. A transcarboxylase subunit of the enzyme then transfers the CO, group to the acetyl CoA. After the formation of malonyl CoA. the next step in the synthesis pathway isthe activation of the substrates by their atachment 10 the pphosphopantetheine group of acyl cartier protein (ACP). The subsequent ‘Steps ate shown in Fig. 42.1 and the overall reaction for the synthesis of palmitate is as follows Sacetyl CoA + MINADPH + 7ATP — palmitate + 8CoA + 6H,0 4 MNADP* + TADP + Pi Fatty acid synthase “Tis enzyme is dimeric with each subunit compose of thee domains 1 Domain 1: acetyl Cos transferase, malonyl CoA transferase seylmalonyl condensing enzyme. 2 Domain 2: ACP, Pkctoacyl reductase, hydroxyacy! dehydrate, enoy! reductase 3 Domain 3: thioester. The dimer is arranged so that the domain | of one ofthe enzymes is facing domains 2 and 3 ofthe other enzyme. Therefor, by rotating on the phosphopantethsinyl group, the activating rections can occur on ‘one ofthe enzymes and the est of the reactions in the eyele ean occu con the opposite enzyme. “The main ste of eool in fay ac syhess isthe enzyme acety CoA earbouylase. The enzyme’s activity is inhibited by hi levels of palmitoyl CoA and the setions of plucagon, andi stimulated by high levels of eitrate and the ations of insti. ‘The assembly of triglyceride the a-glycerol phosphate pathway Triglycerides are formed by the esterification of three fatty acyl CoA ‘molecules with one molecule of d-glyeerol phosphate 1 @-Glycero! phosphate can be formed by the phosphorylation of slyeerol orby the dehydrogenation of dihydroxy acetone phosphate by NADH. 2 Glycerol phosphate combines with wo molecules of faty aeyl COA to form phosphatidate and two molecules of CoA, 13 The phosphate proup is removed to form a dighyoeride. 4 The diglyceride combines with third molecule of fatty aeyl COA to Form a trighyceride and a molecule of CoA. "743 Lipid metabolism II Den ‘Transport of lipids Fig. a: is Bs In this chapter, two principles are discussed: (i) the formation of reducing power for biosyntheses in the form of NADPH: and (ii) the ‘carriage of lipids around the body. PRODUCTION OF NADPH FROM NADP* NADPH differs from NADH only in that there is a phosphate group sattached to C,, bat their uses in metabolism are clearly differentiated, Both molecules can provide reducing power to fuel reactions by theit oxidation to NADP® and NAD®, but the reducing power from the oxidation of NADPH is used to fe biosyntheses while that from NADH is used to fel the reactions involved in the generation of ATP. ‘There are two different metabolic pathways that reduce NADP" 10 NADPH: (i) the pyruvate malate eyele; and (i the pentose phosphate Pathway. Approximately 40% of the NADPH needed for faty acid ‘synthesis is produced by the pyruvate malate cycle (see Fig. 43.1). The remainder is produced by the pentose phosphate pathway. ‘The pentose phosphate pathway “The pentose phosphate pathway is made up of two component pathways {)an oxidative pathway; and (i) a non-oxidative pathway. These both ‘occur in the eytosol. The oxidative pathway is responsible for the formation of NADPH and involves the ineversible conversion of glueose- {6-phosphate, through a series of steps, to ribulose-S-phosphate, Ribulose-Sphosphate isa precursor fora number of important molecules inthe body, which include DNA, RNA, ATP and CoA. If synthesis of these molecules is required in the cell, then Fibulose-S-phosphate will be utilize for that purpose. If ibulose-5-phosphate is not requited for biosyntbeses. the non-oxidative pathway is used. The non-oxidative pathway isa series of reversible carbon skeleton inerconversions which result in the formation of two molecules of| fructose-6-phosphate and one molecule of glyceraldehyde-3-phosphate from three molecules of ribulose-S-phosphate. The starting substrate of ‘the oxidative pathway and the products of the non-oxidative pathway are all metabolites of the glycolytic pathway, and so the pentose phosphate pathway can act asa shunt off from glycolysis, The rate-limiting tep forthe oxidative pathway isthe conversion of ‘slucose-6-phosphate to 6-phosphoglucolactone, This reaction is catalysed by the enzyme glucose-6-phosphate dehydrogenase, whose activity i regulated by levels of NADP*. The controlling factors in the non-oxidative pathway are the availabilities ofthe substrates, Pathophysiology of the pentose phosphate pathway. deficiency of the enzyme glucose-6-phosphate dehydrogenase in red blood cells, is found in 11% of black Americans, This deficiency leads to {decreased levels of NADPH in the red blood cells. NADPH is needed in these cells forthe reduction of oxidized glutathione. Glutathione maintains the reduced state of the cell and is also involved in the metabolism of toxins. The deficiency therefore predisposes the cell todamage which might be prevented with sufficient levels of reduced glutathione Wernicke-Korsakoff syndrome. The enzyme ketolase, of the nnon-oxidative pathway, tightly binds a thiamine pyrophosphate prosthetic group. In the Wernicke-Korsakoff syndrome, the prosthetic group is held less tightly than normal and a deficiency Of thiamine in the diet (e.g. in alcoholies or precipitates characteristic symptoms which include disorienta ‘and deereased mental function. alnourishment) CARRIAGE OF LIPIDS AROUND THE BODY Lipids are transported round the body in structures known as lipo- proteins. These lipoproteins consist ofa shell of polar lipids (phos- pPholipids and fee cholesterol) and apoproteins and a eore of non-polar lipids (triglycerides and cholesterol estes). Carriage of the lipids in this way facilitates their transport in an aqueous medium and enables & very specific targeting system for metabolites to occur. Dietary lipids that have been digested and absorbed follow an exogenous pathway involving chylomicroas and chylomicron remnants. Lipids that have been formed within the body, for example biosynthesis in the liver or membrane turnover, follow an endogenous ‘pathway. This pathway involves four diferent types of lipoprotein which ‘can be differentiated by density and surface protein markers: (i) very low-density lipoprotein (VLDL); i) intermediate-density lipoprotein (ADL): (it) lovsdensity lipoprotein (LDL): iv) high-density Hpoprotein (HDL) “The passage and interconversions of the lipoproteins are shown in Fig. 43.1. As triglycerides are lot from the core ofthe lipoproteins, VLDL become IDL. which lose more triglyeeride to become LDL. [Nascent HDL is released in the liver and acquites further lipid from the mebranesof peripheral cells: ts purpose fs to transfer cholesterol esters {to ther lipoproteins for subsequent hepatic metabolism, Pathophysiology of lipid transport Familial hypercholesterolaemia (type Ha) is caused by a deficiency ‘of the LDL receptor as a result of a number of types of mutation (e.g absent receptor: defective ligand binding by receptor: disruption of transport to cell membrane of receptor). Increased blood levels of LDL. and cholesterol result and these may be deposited in the walls of arteries leading to premature atheroma. Familial hyperlipoproteinaemia (type IID is caused by an sbnocral apoprotcin E and causes inereased bloc levels of remnant particles IDL) with raised triglycerides and cholestrol levels Familial hypertriglyceridaemia (type IV) has an unknown cause, but results i increased blood levels of VLDL with high triglycerides.44 Nucleotide synthesis US nS Pri cine Atos — @ Ko gue arnt eo | yoo i foes | 8 ne reste (MP) i “S N 8 vane (uP) a Nucleotides ste involved in most of the integral biochemical proceases ofthe cell To name oaly few oftheir factions, they are favelied Hidden vorige nd icleiSe CATE), coniialcation GF ribeliir signals tntaclllary (CAMP) and gence information processing (DNA and RNA), The sructure of nucleate is described inch op NS ape pr usanine ‘Guanate = OT tee 8 Ase — B Ni He=0 oH He=O i i i r Hy NH HV O~ cH spare Funarae ms e renee @ «<2 Fee @ | | NS aN Hn—c~" “oH iow oN en) wh te | F Formation by omy teehyerototate PREP Phospharbosy--pyrophesphale EMBLY OF PURINES The purine ring is assembled from five precursors: glycine, glutamine CO, and formyltetrahydrofolte, The Firs step in the sphoribosy-I-pyrophosphate (PRI ribose-5-phosphate und is catalysed by the enzyme ribose ph pyrophosphokinase The second step is the committed step in the synthesis and isthe hospho espartte, inreversible formation of &. hosylamine from PRP. ThisSee nu) Ve Vo = ¢ ae one HCOy * M Fe la ey a eB ema | Neattaneylapanate| | on io fom 9 i 1 ‘00c 1 | 0G UN. os otN, ic” “S=0 we" “C= 0) co, G” “G=0) Pr PAPP i I - | ho sod W 0 reaction is catalysed by amidophosphoribosyl transferase and is driven forward by pyrophosphate hydrolysis, Control of purine ring synthesis The synthesis of the purine ring is controlled mainly at four 1 ribose phosphate pyrophosphokinase is inhibited by AMBP, inosine monophosphate (IMP) and GMP: 2 amidophosphoribosyl transferase is inhibited by AMP, IMP and GMP, 3 the enzyme converting IMP to AMP: and 4 the enzyme converting IMP to xanthylate is inhibited by IMP. xdenylosuccinate is inhibited by Salvage reaetions A common Feature of purine nucleotide synthesis s salvage reactions. The breakdown of nucleotides forms free purine bases, and these can sd by ther direst addition to PRPP. Th AMP) and hypoxanthi (HGPRTase) (whieh forms IMP and GMP), guanine phosphoribosy! transferase ASSEMBLY OF PYRIMIDINES. The synthesis of pyrimidines differs from the synthesis of purines in that the pysimidine ring is synthesized first and is then attached to ribose phosphate, The three enzymes that catalyse the conversion of glutamine to dihydroorotate (synthetase, transcarbamoylase and Alihydroorotase) are domains on one protein. The two enzymes that conversion of fate 10 uridin (UMP) catalyse monophosphate (transferase and decarboxylase) are dom: ‘The committed step in this symthet and carbamoyl phosphate pathway is the formation of carhamoylaspartate from apart [ ore: the term “committed step” mean the firs imeversible stop of the pathway. and is often the step at which the pathway is controlled The enzyme that catalyses this reaction, aspartate transcarbamoylas, is inhibited by eytidine triphosphate (CTP), CTP is formed from UTP and is one of the end products of pyrimidine nucleotide synthesis45 Breakdown of nucleotides Bree Uy op ap — ae ¥ Glanosne ‘oresice MP Paina nuotiephosphoyase | Adana eaninse Glarne«Recee hosts nase __} Nuss {es Pine uci phoschense ating ¢ SARE ONES Foonantin ions phosphate ante dase ie aca radation of purines is uric acid which is The end product forthe d cexereted inthe urine. acids. The enzyme deox uridine triphosphatase (4UTPase) cctalyses the reaction that converts dUTP to GUMP. It is important ‘end products of degradation of pyrimidines that deoxyurdi ‘diphosphate (JUDP) is not present in the cell in se it would become incorporated into DNA The thee enzymes that catalyse the final reactions of pyrimidine ehydrogenase,dihydropyrimidinase high concentrations o breakdown (dihydropy timid and uteidopropionase) can metabolize uracil or thymine and their proceeding intermediates equally. PATHOPHYSIOLOGY Deficiency of adenosine deaminase The defici thought that d _queally inhibits other enzymes involved in nucleotide synthesis. Similar 1 of this enzyme results in immune impairment. It is is a result of substrate accumulation which subse to ths i a defici 1¥ of purine nucleotide phosphorylase which also ‘Gout results from deposition of ric acid crystals in joins (causing severe pin and inflammation) asa result of increased levels of uric acid in the blood. Its often due to a metabotie abnormality which has resulted infan increased production of purine nucleotides. The uric acid may ‘precipitate to form sodium urate crystals which ae deposited in joins and kidneys and cause pain and renal impairment. ‘The biochemical basis for the majority of eases of primary gout is ‘unknown, but the condition is associated with increased uric acid production and/or reduced renal excretion, la rate instances, primary {Rout may result from specific enzyme defects, 1 APRPP synthase enzyme which is insensitive to feedback inhibition bby GDP or ADP. 2A partial deficiency of HGPRTase, The decreased rte of the salvage rection causes increased levels of PRPP which will result nan increase inthe activity of PRPP aminotransferase ‘The symptoms of gout can be relieved by the drug allopurinol, This is metabolized by santhine oxidase to alloxanthine which does'not issocite from the active site ofthe enzyme 9846 Catabolism of amino acids eee Topophan Lysine ine ie - is Phenyaianine = Ostne Fig. ee aeINTRODUCTION Amino acids in the body that are not used in protein synthesis are not stored or exereted. Instead, they are metabolized 1 intermediates which ean be fully oxidized to provide energy or are converted to glucose, fatty acids o ketone bodies. This occurs mainly inthe liver. “The pathway forthe breakdown of most amino acids can be divided imo two distinct phases: (i) the removal of the amino group and its ‘conversion into urea (the Krebs-Henseleit urea eyele): and (i) the ‘conversion ofthe remaining carbon skeleton into pyruvate. one of the ‘metabolites ofthe etre acd eyele, acetyl CoA or acetoaceryl CoA, REMOVAL OF AMINO GROUP AND FORMATION OF UREA eamination of amino acids ‘The removal of the o-amino group from most simino acids is achieved boy transamination reaction. This reaction is catalysed by specific ‘aminotransferases and involves transferring the amino group onto a= etoglutarate wo form glutamate. The loss of the amino group converts the amino acid into an c-keto-acid. The transamination catalysed by ‘alanine aminotransferase is shown below: ‘lunine + c-ketoglutarate —> pyruvate + glutamate “The aminotransferases are dependent on pyridoxal phosphate prosthetic group which is derived from vitamin B, and can accept the ‘amino group and transfer it to c-ketoglutarate. The amino acids serine and threonine are exceptions tothe rule in that their amino groups ean be converted directly to NH,’ by dehydratases. Starvation: role of muscle aminotransferases “The importance ofthe aminotransferases un be seen when the body is subjected to a petiod of starvation, After the glycogen reserves have heen depleteditis vital to maimtain an adequate concentration of glucose in the blood, The body isnot able to conver fatty acids to glucose, so ‘gluconeogenesis must be fuelled by amino acids. The sequence of ‘metabolic events is outlined below. 1 Amino acids of the muscle tissue, especially the branched chain amino scids (valine, leucine and isoleucine), are deaminated, 2. The carbon skeletons ofthe deaminatd a bolized by the enzymes of the citric acid cycle, phosphoenolpyruvate carboxykinase and pyruvate kinase to fort pyruvate 3 Inareaetion catalysed hy alanine aminotransferase, pyruvate can be ino acids are met ‘converted to alanine, 4 Alanine is then released into the blood where it passes to the liver “There itis converted back to pyruvate which can then be metabolized tozlucose. ‘The Krebs-Henseleit urea cycle “The glutamate formed hy the transamination reactions can either be ‘converted to glutamine or to a-ketoglutarate. The conversion 10 _luamine is fuelled by the hydrolysis of a high-energy bond of ATP and is catalysed by glutamine symbhetase, Glutamine can then be used as. fuel source in the intestines or as a regulator of acid-base balance inthe kidney. The conversion of plutamate w -ketoglutarate is catalysed by glutamate dehydrogenase, andthe NH,’ which is formed enters the ‘urea cycle, The formation of oxaloacetate from fumarate provides the ‘urea cycle witha ink to the citric acid cycle as oxaloacetate can condense with acetyl CoA to form citrate. ‘The rate at which the urea cycle functions is controlled by aeety! plutamate which stimulates carbamoyl phosphate synthetase. Acetyl _slutamate is synthesized from glutamate in 4 reaction catalysed by & symthetase enzyme. The activity of this enzyme is inereased by amino acids, particularly arginine. INTEGRATION OF THE KETO-ACID SKELETONS INTO METABOLISM All ofthe amino acids found inthe body are broken down into one or more of seven intermediates in metabolism. They are acetyl CoA, scetoacetyl CoA, pyruvate, c-Ketoglutaate, succinyl CoA, fumarate and oxaloacetate “The amino acids that are degraded purely to cither acetyl CoA or acetoacetyl CoA are termed ketogenic since their breakdown is directed towards the formation of ketone bodies. Amino vids whose breakdown products can be directed towards the formation of glucose are termed ‘lucogenie. The only purely Ketogenie amino acids are te lysine, although isoleucine, tryptophan, phenylalanine and tyrosine ketogenie and glucoger and PATHOPHYSIOLOGY Deficiencies of the urea cycle ‘Any defcicney of the urea eyele is detrimental to the body, since high ‘concentrations of NH, "are toxic andthe urea cyele isthe only metabolic pathway which can convert NH,” to. substance that can beexereted. A total deficiency of one of the enzymes of the cyele results in death shortly after birth, Partial deficiencies result in mental retardation and frequent vomiting Maple syrup disease {In maple syrup disease, there is a deficiency of the branched chain a keto-acid dehydrogenase enzyme. Ths eas to an increase in the levels ‘of isoleucine, leeine, valine and thei 0 keto-acid derivatives in both the blood and urine, The accamulation of these compounds in the urine ‘causes the urine 1 smell similar to maple syrup. Renee the name. IF ‘undetected, maple syrup disease leads to physical and mental retardation, This disease can be detected by adding 2.4-dinitrophenslhydrazine 10.4 urine sample. This compound will combine with a-ketoisocaproate, derivative of lewcin, forming 24-dinitropheny hydrazine. Manag ‘of maple syrup disease involves a specially formulated dit which is low in leucine, valine and isoleucine, Phenylketonuria Phen Iketonutin is caused by a deficiency ofthe phenylalanine mono “oxygenase enzyme, o less commonly ofits tetray drobiopterin cofactor. ‘The disease has an incidence of ound | in 25 0 newhorn babies and show an autosomal recessive inheritance pattern. Inthe healthy state, 78% of phenylalanine is converted into tyrosine which can then be metabolized, and the remainder of the phenylalanine is incorporatedEee eo into proteins. In phenylketonurics, the conversion of phenylalanine into 'yrosine is blocked and Jevels of the amino acd rise drastically in the blond andthe urine. If undetected and untreated inthe early stages, severe ‘complications follow which include abnormal myelination of nerves, hyperreflexia, a low brain weight and mental retardation, These ‘complications are in evidence in untreated I-year-old phen lketonuries who would have seemed normal and healthy at birth, Untreated Piienylketonuria is aso fife-shorening and will usually be fatal before the patient reaches the age of 30 years, The pheny ketonuria gene has heen cloned and sereening programmes have ensured that most phenyiKetonures are now tread The treatment for phenylketonuria isa diet which is low in phenlyaanine, but which sill contains sufieent amount ofthe amino acid for normal growth, nature of the effects of raised level of phenyl: nis sare soon ater bith Because of the se Pernicious anaemia Inpernicious anaemia, there isa deficiency of intrinse fuetor.Insinsic factor i responsible forthe uptake of vitamin B,, also called cobalamin, from the ileum. Cobalamin isa coenzyme associated with two integral enzymes of amino acid degradation, methylmalonyl CoA mutase and eystathione synthase. The mutase enzyme converts methylmalony! CoA, to succinyl CoA and the synthase enzyme converts homocysteine (a metabolite of methionine) to eystathione. The deficiency of the ‘coenzyme results in increased levels of the enzyme substrates methylmanoly! CoA and homoeysteine. Increased levels of meth: malonyl CoA cause acidosis, and increased levels of homocysteine eause homocysteinuria. There are also a number of defects in the metabolism of methylmalonyl CoA which result in acidosis and inereased levels of‘meth lmalonate inthe urine. In approximately S0% of these patients there is a defect in the conversion of a derivative of colbalamin 10 collalamin itself. These patients respond favourably to treatment with, vitamin B,,.. Other patients have a defective enzyme involved in ‘methylmalonyl CoA metabolism, for example methylmalonyl CoA, mutase, nd will not therefore respond to vitamin B, Vitamin B,, is also essential in the synthesis of purines and pyrimidines and lack of cobalamin is damaging tothe hsematopoietic system due tothe rapid tumover of red blood cells47 Synthesis of amino acids Sie THe Men TH Homceytlne nth release aoe ey Adena! ystnione vento tensease romoqyrase tase oysmonsse a eae geen eee ‘hone ramozstne ety cysoce gop 8p: ——— Poe ———p are ae ine espatese are transaminase ‘Serine hydroxymethy enslerase yen Geos Aino ansamiase sen cok rice rneaina Apa 4 Onset >> Cte Gutarine THF = Tearyaotlate Syathetase utamate Asparagine Suesty|CoA ¢— a-ttogaarate Glutamate ehydogenase Gkaamine synnetase clzarae ——— Gitanine | Reductase Pring ¢ SOS pyle 5- ¢ ———= Guana ysomiadehye carbonate (Of the 20 umino acids that exist inthe body, nine cannot be synthesized and are called non-essential amino acids. This chapter will discuss the «have tobe obtained from the diet. These are called essential an synthesis of the non-essential amino acids facids, The remaining 11 ami an be syathesized in the bSUPPLY OF THE AMINO OUP In the synthesis of most amino acids, the amino group is supplied by glutamate. Glutamate is formed in @ reaction catalysed by glutamate dehydrogenase: NH + ocketoglutarate + NADPH +H! — glutamate + NADP*+ HO [A further NH,* group can be incorporated by the amination of slutamate to form glutamine. This reaction is catalysed by glutamine synthase NH,’ + glutamate + ATP — glutamine + ADP + Pi+ H° ‘The amino group on glutamate ean then be transfered to a Keto-acid to form an amino acd by a transamination reaction, Control of glutamine synthase Glutamine synthase is a key controller in nitrogen metabolism Glutamine is involved in the synthesis of a number of important ‘compounds, including the amino acids tryptophan and histidine, the nucleotide CTP and the pyrimidine ring precursor carbamoyl phosphate. The final products of glutamine metabolism bind to the ‘enzyme and inhibit its activity. The activity of ghatamine synthase is also affected by the reversible covalent attachment of AMP toa tyrosine residue in the enzyme. The addition and removal ofthe AMP is catalysed by adenylyl transferase whose activity in tum is modulated by interchangeable regulator proteins, When levels of ATP of « Ketoglutarate are raised, AMP is removed from glutamine synthase, Which increases its activity. When levels of glutamate or glutami raised, the AMP is added to the enzyme, which reduces is activity. Synthesis ofthe carbon skeletons "The 11 non-essential amino acids that can be synthesized by the body ace alanine, arginine, asparagine, aspartate, cysteine, glutamine, tlyeine, proline serine, glutamate and tyrosine. The formation of all ofthese amino acids except for tyrosine is shown in Fig. 47.1. Tyrosine is formed by the hydroxylation of phenylalanine by phenyl hydroxylase phenylalanine + 0, + tetrahydrobioptes tyrosine +10, + dihydrobiopterin ‘Tetrahydrobiopterin acts as an electon carrer inthis reaction and {s regenerated by the reduction of dinydrobiopterin, which uses NADPH asa reductant Serine, lyeine and eysteine are formed from an intermediate of the lyeolytic pathway 3-phosphoglycerate. The phosphatase enzyme which converts phosphoserine to serine is inhibited by serine, providing @ feedback regulation forthe pathway. ‘Glycine is formed from serine by the removal of u ene-eazbon unit and its attachment toa tetrahydofolate (THF) cartier. The carbon unit is carried on either the N, of N,, nitrogen atom of THF as methylene- ‘THE. THE can exist in 2 number of oxidation states depending on the ‘carbon group carried, for example a methyl-THF donates methyl group. {to homocysteine to form methionine. Anexample of another carer of earbon units isadenosylmethionine. “This cartier has a much higher transfer potential for the release of the ‘carbon unit than has rethyl-THE, and is used to methylate a numberof important molecules, e.g. noradrenaline. The formation of adenosyl- ‘methionine forms part of a methionine salvage pathway in which ‘methionine is regenerated by the methylation of homocysteine by rmethy/-THE. The enzyme that catalyses the formation of methionine from homocysteine is a methylransferase which uses vitamin Bas 8 ‘coenzyme. ‘Cleavage of eysathione results in the formation of the amino acs ‘steine and homoserine, Homoserne is then converted ino ketobutyrate ‘which i transported into the mitochoruria and metabolized to suecinyl Coa.Fie. 4a 48 Integration of metabolism Feeding and starvation re associated with changes inthe metabolism. of the body. Feeding results in a large influx of metabolic substrates into the body and the metabolism has to adapt quickly to store the nutsients as proteins, lipids and glycogen. Starvation is exactly the ‘opposite of feeding and the metabolism of the body must be able to co-ordinate a controlled breakdown of the body’s energy storage polymers to provide adequate substrates for enerzy generation FEEDING Shortly after feeding, insulin is released into the circulation from [Becells in the islets of Langerhans ofthe pancreas."The overall effect of insulin on metabolism is the stimulation of biosynthetic pathways so thatthe digested and absorbed substrates such as glucose or aminoacids can be converted into a form that can be stored. Some ofthe eflexts of insulin oa metabolism have been described in earlier chapters and its ‘overall effects are shown in Fig. 48.1. STARVATION “There ate three distnet phases through which the metabolism of the body passes when in a state of starvation: (i) the post absorptive phase: iy the gluconeogenic phase; and (ii) the ketotic phase. These three ‘phases nd their associated metabolic states are shown in Fig. 48.2.Dear) Post absorptive phase | _Lowbled aba (about2 to 4 hours) “Tidyerde reakconn in acipose sv and cogent ine her oleaso of gucose and fat acd ino thebiecd Gluconeogenic phase (24hours) | fue Bowe aid ucagen Increased aly ac oxdaton nthe er Increased lel ATP, atl Coh and NADH Povey encanta pata ene i aa Soon Scent nacmneneetato peta Ketotic phase ‘Ketone bodies caus lease of isin ich preenis extensive protsn ycoyis Fie, S a2 ‘The ketotc phase lasts only a long a th cain be broken down to form acetyl CoA for ketone body formation, of amine acids from protein breakdown, Death eventually occurs from Oxaloacetat, derived from glucose, is supplies in the body ketone bodies. The glucose isin turn, derived from the gluconeogenesis «for the metabolism of the extensive breakdown of functional proteins49 Gas transport Gas transport Pa kPa Pa 5 0 8 Fe 5 0 10 § sel os Fetal He : pir (He =" Venus bod aenogiotn tH) pH7 2 causes anyge-inng Opreease ane (rom Ho Nena 2 100 100 50100 Pop mnty thas 75mm Carbon dioxide transport mm = 0.198 a aeHCOS 7% Wa Pa bound Ho 1% § 0 5 10 ‘dso plasm m% yoalbin § son fn poses : axa 56 ia ae P0829 une psp ayers a © 00 % w Poometg Pogmty Fig on s TRANSPORT OF 0, AND CO, In blood, most O, is hound to Hb. When ©, is bound, Hb is called ‘oxyHb. Fb is aed protein present at about 150 gf, Each gram of Hb binds about 1.36 ml of O,, ie. lf blood can carry about 20 ml O, ‘The O, saturation curve for Hb is sigmoid. Aumosghow: Pop = 21KPa (159mg) > ota Figs | | Hoo Lung: Po = 1a (100mm) ie Hoy | ‘si HOO + HHO: > Hii + Op PO, erties of the curve The steep part of the curve lies within the range of physiological extrapulmonary P ‘small fallin issue Po, will result in a large dissociation of O, from Hb. The si ss O, is taken up it values. Therefore noidal curve is ‘consistent with the oecurrence of co-operativitysinereases the afinity of Hb for more O,. Ata Po, of 3.6 KPa, Hb is 0% saturated with O, (the PA high P,, means alow affinity for O.. The Hill cooffcient for Hb is 2.8. This indicates positive eo-operatvity Asiall shift ofthe eurve to the right meansa sharp lons of O, olding anda small shift tothe left implies a sharp ality increase. Hb binds (©, weakly at low Po, and tightly at high Po, Hib allosterism “The binding of O, 19 Hb causes « homotrophie effect, when a ligand changes the afinity of another protomer of protein for the same ligand (0,). Hb also shows heterotrophic interactions, i. the affinity for ©, isdeerease by the binding of other ligands. Tce ligands that deerease the affinity of Hb for 0, are CO, H’ ions and 2.3-bisphosphoglyeerate (2.3-BPG), which occurs in red blood cells, and its net formation is. a diversion from glycolysis, €O, and 1 ‘CO, and H’ bind to Hh, decreasing it affinity for O,- Conversely, when , binds to Hb. it lowers Hip affinity for CO, and H* ions, and both Aissociate more easily from the protein. This interaction between O, (CO, and H’ with Hb isthe Bohr effect. In metabolically active tissues CO, and H* concentrations are high. These bind to Hh causing the release of O, to the tissues, Hb carrying CO, and HY is transported to ‘the lung, where O,is taken up by free © -binding sites on Hb, and CO, and H’ fons are released ‘Transport of CO, ‘CO, diffuses from the tissues into the blood, and into the red. cells, where itis converted into catbonie aid by carbonie anhydrase CO, +HO=H,CO, HCO, +H The reaction is driven tothe right because CO, i continuously entering the red blood eel. H* ions bind to Hb (releasing 0.) and FICO, diffuses ‘down a concentration gradient into te plasma in exchange for CP. Thus, ‘auch CO, travels tothe lungs as hieronate, Some CO, binds reversibly ‘ounionized amino acid moieties on Hb promoters, forming negatively charged earhamino groups, The earbamino groups form salt bridges ‘with groups on Hb which are positively charged, thereby promoting Stability of deoxygenated Hb. ‘Transport of H* ions Hons generated in the blood must be buffered, to prevent acidosis, and H" binds to ionizable groups on Ho globin chains. Thus, Hb acts 3s, 1 baller. H* ions bind mainly to the imidazole group of C-terminal histidine esidues on Hib Bechains. When O, dissociates from Hl, the protein polypeptide chains alle shape, bringing imidazole groups of the histidine residues elose to COO" groups of aspartate residues, and these form @ non-covalent electrostatic bond which stabilizes the deoxygenated Hb, When the pH of blood falls this shifisthe O, saturation {curve to the right, and more O, is eleased, thus increasing the bering capacity of Hb, Tm the lungs, Hb binds O, and changes shape. H’ is released, and ‘oxyHb has a negative charge. This is balanced by the positive charge of ions inthe red blood cel. In other words, oxyHb acts asa stronger acid than Hb, H- combines with bicarbonate to form carbonic acid, Thus the concentration of bicarbonate ions in the red blood cell is reduced, and bicarbonate ions diffuse down a concentration gradient from the plasma int the cel. To maintain electical neutrality in the cell, CI-ions diffuse out of the red blood cells into the plasma. This movement of Ch ions is termed the chloride shift In the tissues, O, dissociates from Hb and diffuses into the cells, Hb takes up H’ from carbonic acid, and bicarbonate fons are formed ‘The negative charges of red cell bicarbonate ions are balanced by K* ions. Bicarbonate ions diffuse from the red cell into the plasma. and Cl- ions diffuse from plasma into the red blood cells in exchange for bicarbonate ions. CO, enters the red blood cells and reacts with H,O to form carbonic acid, Therefore. the Pco, within the red blood cell is kept low, and this ereates a concentration aradient allowing more CO, to diffuse into the red blood cell from the tissues. 2.3-BPG is present in the red blood cell in concentrations equivalent to those of Hb, 2.3-BPG binds to Hb, and shifts the O, saturation courve tothe right, When 2,3-BPG is not bound, Hb is saturated with (0, atthe Po, of the tissues, and litle is given up to them. 2.3-BPG is negatively charged, and binds to postive charges on the B-globin chains ofthe deoxygenated form of Hb, and slots into aeavity between the two chains. In the lungs, as Hb binds more O,, the protein shape changes so thatthe B-globia chains move closer together and compress the sites where 2.3-BPG is ound. FETAL Hb (HF) The fetus derives O, from the maternal circulation, and HDF has & higher affinity than does adult Hb (HbA) for O,. HBF does not have Brehains, but ychains, which possess fewer positive charges in the cavity where 2.3-BPG is bound. Therefore, HBF binds 2,3-BPG less tightly than does HbA. ros50 Haemoglobin ke goin sero genes on cromosome 1 (haan) © » 2 Fig, 504 B-GLOBIN FAMILY Several different forms of felike globins are formed during embryo. genesis and adult fife in humans another vertebrates In humans, these include B- &, A. G- and e-globins. The genes coding for the Polypeptide chains occur reatively close together on chromosome 11, and express chains whieh are very similar in sequence identity. Only the Band 6-chains continue tobe expressed after 6 months of postnatal Sie in humans. STRUCTURE OF Hb Hb consists of Four subunits. Each isa globin molecule containing & haem group, und the Four subunits are held together by non-covalentbonds. HbA consists of two ce-chains, each 141 amino acids long, and two Bechains, each 146 amino acids long. There is a high degree of "uniformity in tertiary structure among the diferent subunits of Hb, and ‘each chain is arranged in multiple @-helix regions interrupted by turns ‘of the polypeptide chain that Force the subunit into a spherial shape. THE HAEM GROUP Each subunit of Hb contains a prosthetic haem group. Haem consists of| 2 polyringed porphyrin molecule, with an atom of iron (Fe) in the ferrous (Fe(L)) format its centre. Hb and myoglobin porphyrin is termed protoporphyrin IX. The Fe is bound to the four N atoms of the haem, bats situated outside the plane ofthe molecule beeause iis larger than the space between the N atoms. The Fe is also bound 10 the peptide chain of the subunit through a inkage with one of the histidine (His) residues. ‘Combination of Hb with O, (©, can combine reversibly with each of the four subunits of Ht. The O, binds to the Fe atom on the face ofthe haem group opposite to that ‘bound to the protein. The frst O, binds to Fe in an desubunit. This, ‘overeomes a repulsion between a His residue and the porphyrin ring and allows Fe to move into the plane of the ring. AC the same time, several ofthe ionic bonds linking amino acid residues rupture. This ‘causes a conformational change over the whole Hb molecule, The two Besubunits move closer to each other, although they do not actually touch, This forees the dissociation of 2,3-BPG. As the B-subunits move closer together, the two @-chains move further apart, and this makes it easier for O, molecules to gain access to the haem sites, Thus, as succeeding O, molecules bind, so they open o relax’ the Ib molecule further, and this increases the apparent afinity of the molecule for O, the fourth O, molecule finds easiest to bind, i. has the highest apparent affinity for Hb MYOGLOBIN Myoglobin isa protein in muscle cells. Like Hb, itbinds O, but, unlike Hb, myoglobin consists of a single polypeptide chain, 153 amino acids Jong, with one haem group which binds only one molecule of O,. Its saturation curve is therefore not sigmoidal Functionally, myoglobin provides a binding site for O, that ean be ulized by the metabolically very aetive skeletal and canfiae muscle Fibres. Together, these two tissues account for about 30% of the consumption of the human body at rest. The saturation curve fe myoglobin, over most ofits length Hes fo the left ofthat for Hb In other words. it has a higher affinity for O, than has Hb, Therefore, in the tissues such as muscle, where the Po, is about 2.5-3.5 kPa, when HD is Jess than 50% saturated with O,, myoglobin is fally saturated. When the tissues demand more O,, the Po, drops to less than 0.2 KPa, and at this order of magnitude, myoglobin releases most ofits bound O,, Ualike Hb, the binding of ©, by myoglobin is not influenced by 2,3-BPG concentrations, CO, oF H PATHOPHYSIOLOGY OF Hb Fi is associated with several disease states, Quantitative determinants, Patients may present with aod blood ell RBC) count inthe normal range, bat with a mein cel volume (MCV) outside the normal range (microcstsis),abnoemal mean cell Hb content (MCH) and abnormal scan cel Hh concentration (MCHC: hypochromia) This is microeytc anaemia, although in ther forms ofeaemia the patient may preset with a lowered RBC count “Te anaemia may be caused by a dltry shortage ofthe Fe reed forhaem eyes, Individuals at iskinclude women whoare pregnant menstruating or lstatng and vegeterins, especially yepans Miroyti anemia my result roma fale t ize Fe Indvidans ats include thse exposed to tonic substances such a ea which tock -SH eroups of enzymes tha catalyse reactions involved in the synthesis of haem. ‘Chemicals can cause a quantitative reduction in avilable Hb, For example, inhalation of carbon monoxide (CO), canbe fatal. The CO forms a sabe, bight red complex with the haem group and doesnot dissociate, Another example isthe oxidation ofthe Fel) to Fel}, to form methemoglobin. Methaemoalobin may be formed through hereditary defects globin synthesis, or trough oxidizing compounds Inthe Fell tates, O, i not hound. and when present ogee with somal HbA methaemazibinshitsthe Ostraton curve ofthe HDA tothe let making is bound O, less accessible othe tissues (Qualitative determinants In sickle cell anaemia patients synthesize MS, an aberrant form of HA. in which a non-polar valine Val) residue replaces a polar glutamate residue at postion six on the B-subunit (Giv,.). Gi, les on the outer surface of HbA. and inthe deoxy state of Hb, the non-polar Val residue forms hydrophobic bonds with other HbS molecules, which causes ‘polymerization and precipitation of Hb inside the RBC. Consequently ‘the RBC acsumes a characteristic sickle shape and loses elasticity, which results in blockage of the microcirculation by the cells. 105,51 Molecular chaperones Fis. su Molecular chaperones regulate the folding of many newly synthesized proteins. The chaperones bind newly synthesized proteins, preventing them from aggregating inthe eell, and mediate protein folding into their native state. There are several, structurally uneelated families of molecular chaperones, The term “molecular chaperone” was originally applied to the proteins nucleoplasmin and the chloroplast biphoxpbate carboxylase, which promote the assembly of nucleosomes, “The molecular chaperones can organize themselves into structures, called haperonins, which contain a central cavity in which an unfolded proven is shielded from the cellPRINCIPLES OF CHAPERONE ACTION ‘Molecular chaperones bind and stabilize proteins in their n fem, and release them in such a way a8 to facilitate their folding ‘They can recognize structural features of proteins in their unfolded form. in particular certain amino acid sequences, but cannot bind folded proteins, "Newly formed proteins extend from the polysomes, amino-terminas leading, as unfolded peptide chains. These cannot sta folding until a critical length has been reached: about 100 amino acids, These form a protein domain, which facilitates he further folding ofthe entire protein inaco-operative manner. When the chain is elongated, its hydrophobic residues are repelled by the aqueous environment and tend to bury themselves within the protein, Since so maay different proteins are being. synthesized simultaneously hydrophobic residues from different chains ‘ould interact to form aggregates, and this has been shown to huppen in vitrin the absence of molecular chaperones, Molecular chaperones protect the nascent protein from these interactions by shielding hydrophobic surfaces during peptide elongation, and possibly also during the folding process and during. translocation of the protein across membranes, and by binding the ‘completely synthesized, but as yet unfolded protein, and allowing it to fold unhindered by the other cellular processes going on around usp HSP60 and HSP70 ‘The principle of molecular chaperones may provide a clue as to why LSP are produced during cellular injury or disease. tis known that the 'HSP70 proxeins can bind proseins after they have been incompletely denatured during cellular distress, Therefore, they may provide a means ‘of protecting the cell from undergoing massive protein aggregation LHSP70 proteins comprise o functional domains: (i) a polypeptide- binding domain atthe carboxy terminal: and (i) a nucleotide-binding site atthe amino terminal. There is evidence that ATP is bound and hydrolysed to ADP at the ansino terminal of HSP70, and this provides the eneray to induce a conformational change in HSP70 that results in the release of the fokded polypeptide. Action of HSP70 ‘The mechanism whereby HSP7O proteins act in concert to regulate proven foding and rolease in Escherichia col cytosol has been partially elucidated. There are at least five proteins in the cytosol that mediate protein folding, namely DnaK, DnaJ, Grp, GroEL and Grok. They regulate the folding of newly synthesized proteins, and may also protect already folded proteins during periods of cellular stress. They have ready been shovsn to inhibit the heat denaturation of a heat-labile protein, luciferase, produced by fireflies. The steps in protein folding ar the following. 1 Dnal and Daak, with bound ATP attached toi, bind the unfolded ‘polypeptide chain as itis formed bythe ribosome, and the bound ATP. is hydrolysed to ADP. 2 Dnal and Dna bind t each other asthe polypeptide starts to Fold, and the Daak-ADP-Dnal-polypeptide complex is stabilize. 3 The protein Grp promotes the dissociation of ADP from DnaK, pertaps hy binding to the site where ADP is bound. ATP binds witssite ‘on Dnak, and the folded polypeptide is released. 4 Insome cases, the released protein bound by GroEL, which, through an ATP-dependent interaction with GroES, permits protein folding to be completed. CHAPERONINS “The term ‘chaperonins” has been given to the HSP60 proicin family ‘The term describes a quatemary structure of proteins which assemble 1 form a compartment in which nevly synthesized proteins can fold, protected from other folding proteins, hus eliminating the risk of protein aggregation. Analysis of chaperonins has revealed structures consisting ‘of 14 subunits, stacked in wo heptameric sings with a central cavity, Which ean hold proteins of up to about 90 KDa in size. In. coli, the ‘chaperonins are made up of two HSP60 proteins, GroBL. and Grok, ‘The newly synthesized proteins are held in the chaperonin in what has heen called “molten globule" state. The term deseribes partially folded proteins whose hydrophobic surfaces have not yet become totally buried inthe protein, GroEL and GroES interact viaa sequence of ATP binding ‘and hydrolysis, which provide the energy fr the binding and release of the folded protein, Eukaryotic chaperones and chaperonins In yeast cells, molecular chaperones have been described whose function isto ascisi the transfer of proteins across the mitochondrial membrane. Proteins formed in the cytosol must fist be unfolded before they ean ‘ross the mitochondrial membrane, and refolded once they have done so. This is accomplished through the action of mitochondrial molecular ‘chaperones. There is evidence that proteins inthe eytosol are held in an unfolded state by HSP70 proteins, and the enengy for this is provided by ATP. The polypeptide chain passes through the lipid bilayer ofthe mitochondrial membrane, and onthe inner surface the polypeptide chain is folded under the diretion of HSP70, acting together with HSP6O. ‘A numberof ther chaperonins have now been described, including ‘one found in eukaryotic eytosos, called TCP-1, or CCT (also called cchaperonin of eukaryotic cytosol), which is particularly abundant in ‘developing embryos, testis and lymphoid tissve.Ithas been shown that disruption af yeast chaperonins are lethal for the cell vorAbbreviations cDNA cor. GMP coRP CH.COOH CH.COONa adenine acyl carte protein tdrenocoicotrpin adenosine diphosphate Adenosine mouaphephie adenosine triphosphate Adenosine triphosphatase tase pairs 23-tisphosphoglyeerate cytosine calcium calcium chloride cyclic AMP Cataboie activator protein choleystokinin complementary DNA tine triphosphate ‘lic gumosine monophosphate calcitonin gene-elated petide acetic acid sodivm acetate chlorine carton monoxide carbon dioxide coenryme A carboxyl group cytokine receporsuperfaity extn phosphate cytocrome Fdeoxyibo- daiton diacyelyceot deoxyeytidine triphosphate dideonynucteatde A sopropynhoaphottordate Secciyrotestosterone deoxyribomilee acid deoxyribonulease deoxythymidine phosphate dey diphowphate deoxyuriin triphosphatase excel id longation factor pidermal gro ctor clectromative force endoplasmic reticulum favine adenine dinileoide fbcobas growth factor formyl methionine uanosine diphosphate ‘growth hormone fanosine monophosphate HDL. hie He HGPRTase HIV moRNA HIRE. HSP Hyp 1 1cF DL. iF Tea IGF Mel IMP P, IRS JAK, K Ky LDL. une MAPK MCH Mev Me MGE MCHC guanosine triphosphate _2uanosine triphosphatase hydrogen hydrogen carbonate hydrogen peroxide phosphoric acid haemoglobin adult Hb fetal Hb high-density lipoprotein high frequency of recombinatio mercury hypoxanthine-guanine phosphoribosyl transferase human immunodeficiency virus heterogeneous nuclear RNA hormone response elements heat shoek provein hydroxyproline inosinie acid intracellular Duc intermediate density lipoprotein ation factor ymunoglobulia A insulin-like growth Factor ierleukin | snosine monophosphate Inositol trisphosphate insulin receptor substrate | Sanus kinase potassium Michaelis constant low-density lipoprotein Jong terminal repeats mitogen-activated protein kinase ‘mean cell Hb content ‘mean cell volume ‘magnesium mobile genetic element mean corpuscular Hb concentration ‘mouse mammary tumour virus ‘manganese messenger RNA sodium nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate ‘amino group oxygen hydroxy! group Phosphorus-32 pascal polymerase chain reaction platelet-derived growth factorpPhosphofructokinase inorganic phosphate ‘phosphatidlinosto!-4.5-biphosphate protein kinase C phospholipase A, phospholipase C pyridoxal phosphate ‘S-phosphoribosyl-I-pyrophasphate sas constant red blood cell release factor ribonucleic acd ribonuclease ribosomal RNA, resistance transfer factor Svedberg uit small eytoplasmie RNA small nuclear RNA signal recognition panicle signal transducers and transcription aetvators thymine transforming grow factor tetrahydroflate {hymane monophosphate transfor RNA thymine pyrophosphate rai tundine diphosphate ump ‘uridine monophosphate ur ‘uridine triphosphate uw ultraviolet VLDL very low-density lipoprotein Zn zine Amino acid abbreviations Ala alanine Arg arginine Asn asparagine Asp aspartate cys cysteine Gin glutamine Glu slutamate Gly lyeine His le Lew Lys lysine Met methionine Phe phenylalanine Pro proline Ser serine The threonine 1 tryptophan yw tyrosine val valineGlossary site ribosomal recognition ste where next mRNA codon is exposed to incoming (RNA activation energy critical energy level for em active transport energy-requiring movement of substances across biomembranes affinity strength of attraction between wo binding sites ‘agonist Tivand that riggers a response (sce ligand) allele alternative form ofa gene that an occupy a chromosomal genetic locus allosteric proteins can alter binding ste properties in response to ligand ‘occupancy at another site amphipathic molecule possessing both hydrophilic and hydrophobic properties anaemic deficient in haemoglobin anion negatively charged ion annealing ‘wo complementary nucleic acid strands joining antibody immunoglobulin produced in response to an antigen, which bonds it anticodon set of three consecutive RNA bases complementary to @ ‘mRNA codon antigen substance, usually foreign to body. that provokes antibody formation antimetabolite substance blocking # metabolic (usually eneyme) antiporter membrane protein transporting substance across mem= brane with simultaneous transport of another substance the opposite al reaction to occur Autocrine hormonal ation on call autosomal recessive Mendelian recessive genetic inheritance carried ‘onan autosome autesome chromosome other than sex chro avian referring to bieds Dbucteriophage virus that infects hactria base proton (HI*) acceptor in solution eg. purines, pyrimidines ‘benign medically, « non-cancerous tumous that des not invade other tissues or destroy any healthy tissue C terminal free -COO group at end of « polypeptide chain cap-binding protein binds to cap region of mRNA; required for initiation of eukaryotic wanseription ‘careimogen agent that may cause cancer ‘eardiotonie heart stimulant cation positively charged ion ‘DNA library collection of DNA strands complementary to source tissue DNA ‘chi sequence repeated short DNA sequence on bacte where RecA-mediated recombination is stimulated ‘hondroblast cartilage cell producing cartilage matrix loning producing idenica cells or molecules from single starting cell ‘or molecule ‘co-operative binding ligand binds to protein and changes affinity for ‘other sites for the same ligand on the protein codon set of three consecutive hases on DNA or RNA, specifying an by substance produced by the same ho amino aid ora signal for the end of translation ‘coenzyme (cofactor) non-protein required by a protein for bioactivity cofactor see evenzyme conformation thrce-dimensional arrangement ‘constitutive gene a gene continuously expressed withey transcription initiation factor ‘control element a DNA sequence that influences the expression of nearby genes. ‘cosmid. a cloning vector plasmid containing phage A cos sequences ‘cotransport linked transport of substances across a biomembrane cerista mitochondrial inner membrane infolding crossover exchange of genes between homologous chromosomes during meiosis eytoskeleton internal protein skeleton of eukaryotic cell eytosol soluble eytoplasmie compartment degenerate one amino acid can be encoded by several different codons dimer protein formed of two subunits diploid organism whose cell has two sets of chromosomes, ocking protein protein that ‘places’ other in its binding site domain structurally defined membrane or chromatin region, or globular region ofa protein electrochemical gradient transmembrane gradient defined by ionic and electrical gradients ‘endocrine ductiess glandular function ‘enhancer DNA contol stein eukaryotic genes, whose activation by specific proteins increases transcription of the gene ‘eukaryote organism whose cells have bounded nvclei containing organized chromatin and cytoskeletons ‘exocytosis secretion of chemicals from eukaryotic cells ‘exon DNA sequence unit coding for par of a polypeptide, or for FRNA or tRNA, facilitated diffusion membrane transport down a concentration pradient utilizing a carrer system, but no energy familial eaits Found in some Families; not necessarily inherited {Abril thread-tike component of fibre fibroblast connective tissue cell fip-Nop ‘transition of lipid or protein from one membrane surface to the other G protein guanine nucteotige-binding protein on eytoplasmic surface Forming part of hormone signalling to target cell genetic recombination meiotic exchange of DNA between homolo- ‘gous chromosomes during gamete formation in sexually reproducing organisms ‘genome genetic database ofa single organism or cell ‘genomic library collection of DNA cliromosomsal fragments from one genome ‘globin protein constituent of haemoglobin slucocorticolds adrenal steroids influencing some are ant-inflammatory sllycosides compounds which when hydrolysed yield a sugar and anon sugar, eg. digoxin slycosylation formation of eukaryotic glycoproteins through addition ‘of oligosaccharide side chain to protein the need for ‘of cell memb carbohydrate metabolism:growing fork locus in DNA replication hhaploid cell or organisin having one set of chromosomes helical spiral ebain arrangement of protein or nucleic acid molecules Forming rod-ike helix hepatocyte liver cell hheterogencous having dissimilar components heterotrophic effects allosicric effects due to interactions between sfferent ligands ‘homodimer protein consisting of two identical subunits ‘homologous resembling in origin and structure homotrophic effects alloscre effects due to interactions between identical ligands hormone response element (HRE) hhormone-receptor complex hhydrolysis addition of OH. and H’ ions of H,0 toa molecule which is ‘consequently split into simpler molecules hydrophilic water aracting hydrophobie water repelling in vitro in tes tubes (in glass’) region of DNA that binds inducer chemical or physical stimulus to gene expression or enzyme action sercalate (0 slip bercen adjacent bases in DNA. Intron non-coding region of DNA that may be transcribed but later spliced out of MRNA Isoelectric pH pH at which protein is uncharged isomer compound chemically identical to others, but with different spatial arrangement, 2. stereoisomer Klenow fragment fragment of DNA polymerase I, containing all the ¥- 5 exonuclease and polymerase activity lup-joint intermediate hybrid in genetic recombination ligand a molecule that binds to another with functional purpose lipids clas of hydrophobic compounds including fas, phospholipids ind steroids lipoprotein compound composed of protein and lipid lumen intemal space of subcellular organelle or of sac-ike or tubular ‘malignant tumour that destroys tissue of origin or invades and destroys other tissues ‘matrix medium or ground substance of tissues ‘melosis nuclear division producing haploid daughter cells from diploid parent cell metastasis spreat of cancerous eels to other tissues micelle spherical ordered arrangement of molecules such as phospholipids in aqueous medium mitosis nuclear division, when daughter cells have identical ‘chromosomal complement as parent mobile genetic element DNA sequence able to be inserted on same or ‘other chromosomes and alter gene expression (also called transposable element) monomer molecule composed of a sing rmutagenie able to cause a mutation tation change in natute or composition of DNA resulting in change in characteristics of gene expression in cell [N terminal amino terminus of polypeptide chain nascent newly synthesized: not yet active nonsense codon termination codon ‘Okazaki fragments short DNA sequences formed on lagging (> 5) strand during discontinuous DNA replication ‘oligomer moleeule composed of afew monomer units ‘oncogene gene curried by cancer cell or virus, that is partly or wholly responsible for tumour formation ‘operon prokaryotic genetic unit in which several genes are clustered ‘and transcribed into polycistronic mRNA ‘organelle functional structure within 9 eukaryotic cell ‘osteoblast bone-forming cell site ribosomal site where the list mRNA codon was read (see also A site) palindrome sequence res ‘AACAA paracrine local hormone acting on neighbouring cells passive transport simple diffusion down a concentration gradient includes facilitated diffusion pentose a monosaccharide with formula (CHO), ribose phage see bacteriophage ‘phasmid. vector consisting of « combination of a plasmid with phage 2 phenotype biochemical and physical characterstes of an organism pinoeytosis uptake of liquid into the cell plasmid circular actrial oF yeast DNA replicating independently of chromosomes polyeistronic mRNA. codes for more than one polypeptide polymer macromolecule consisting of several similar or identical subunits polysaccharide macromolecular carbohydrate polymers, © glycogen polysome ribosomal aggregate on mRNA during translation primer short fagment of RNA necessary to initiate DNA polymerase ation primosome prepriming protein assembly necessary for primer synthesis prohormone hormone precursor prokaryote unicellular organisms, e.g bacteria, lacking membrane ‘bounded nucleus and other organelles such as mitochondria promoter DNA sequence necessary fo iitiatio proofreading. property of DNA polymerase to detect base mismatches prosthetic group non-protein moiety. e.g. haem, forming part of protein protomer inactive enzyme form proton Hi (H,O" in some texts) purine base, commonly adenine or guanine in nucleic acids Pyrimidine base, commonly eytosne, thymine or uracil in nucleic acids receptor protein that recognizes ligand, and tha ‘primary member in the chain of communication between ligand and call receptor antagonist ligand shat binds receptor, blocks agonist binding and produces no response receptor superfamily. group of intracellular receptors, with structural Similarities, which at as transcription activators, e., glucocorticoid and retinoic acid seveptors recombination exchange of DNA between homologous chromosomes during meiosis replication fork Y-shaped point where DNA is unwound and simultancously replicated replicon unit of DNA that replicates sequentially and eontains an origin of replication replisome proicin assembly on DNA, nceded for replication repressor bacterial protein that binds to the operon to repress transcription resistance transfer factor (RTF) factor on plasmid that enables transfer of drug resistance to another bacterial cell ing the same both ways, e.g. in DNA: of transcription constitutes the uMsecond messenger intacelllar chemical signal symbhesizedin response {stimulation of receptor on cell membrane ‘sedimentation coefficient estimate of size o ‘ate of sedimentation in a suerose gradient semi-conservative replication means of DNA replication ‘sex pilus means of transmitting F factors from one bacterium to another ‘Shine-Dalgarno sequence bacterial mRNA S’-AGGAGG-3' sequence before initiation codon signal sequence temporary hylrphobic sequence of amino acd at amino terminal, important fr transfer of secretory product across membranes SOS response sequence of repair responses in E.coli in response to damage stimuli splicing. cutting nucleic acid inorder insert sequences, thus creating recombinant nucleic seid splicasome assembly of ribonucleoprotein that splices RNA, stop signal signal that stops macromolecular elongation supercoiling (isting of the double helix on itselt symport simultaneous, linked transport of two molecules across & somolecule from its biomembrane, both in the same direction template patter ftom whicha molecules symhesized..g. DNA strand isa template for its repic transcription synthesis of complementary RNA from DNA, transition DNA base substitution causing mutation translation protein synthesis by ribosomes on mRNA template translocation removal of part of « eheomosom homologous chromosome transposable element sce mobile genetic element transposition replication of a DNA sequence on one chromosome in ‘another chromosome transposon replicated tansposed sequence {transversion substitution ofa pyrimidine fora purine or vice versa Uniport_ membrane transport of a molecule by & membrane prot van der Waals forces weak form of non-covalent bonding between ‘neural molecules vector agent that carries a message, e.g. plasmid, bacteriophage terion ion with negative and positive charges, e.g. amino acids Wo another, non:Index Page references 1 igues appa in ale type ant hose Te ables pear i bald type scetylholine, 11 acid-base balance, 59 ‘dbase imbalance, $9 ‘is and bes, 86-57 ‘ctinomyein D 25 sce tampon. 7 ‘slenosine deaminase deficiency, 92 ‘xenosylmethionine, 99 ‘aenylate cyelse, 9 ‘enyly ansferse, 99 ‘realine, 65, 87 ‘srenocoricotopin, 87 Sgonists, 1 alanine. 99 allosteric sites, 70 silstriam, 71 ‘Ames test, 19 midophosphoribosy ranserae. 91 fino acs, 18 euabolsm, 94-98, 96-97 integration of ket-acd skeletons, 95, removal of amino group, 95. deamination. 95 sential, 9 pathophysiology. 98-97 yess, 94-59 2-amino-phosphonovalerate, 11 ‘aminoaey-ARNA syntbetse, 30,31 sminoacaton rection. 31 Spite ips, amylopectin, 75 drogen resistance, ‘ions 35. ankyrin, $ ‘agonists, 11 antibiotics, 2938.9) codon pit, 23 antiport. 7 gine, 9 “sparagine, 99 ‘pre. 99 satenator sites, 25 sutoutvaion, 75 utcatalyis 75 ‘uterine signaling, 8 B protein, 27 bacterial voi, 39 bacteriophages, 21, 55 bse paing. 13 solar transporter, 73 bie salts, 77 bod buffering tne, 39 bute. 57 CAAT bo. 27 ‘ator #6 ‘akitonin gene-related peptide, 46 ‘alum pump. 7 ‘almodolin, 7 ‘cancer, 24-9, 50,31 ‘malgnaney, $0-81 atbamoyl phosphate, 9 carbohydrates, digestion, 74.75 ‘bon oni anspor, 103, ‘atabolte activator protein, 48 atbolte repression, 43 catalytic ad, 67 ations, 56 ‘lla chemistry, 22 ‘ephalin, 8 ‘eramide, 5 haperonins, 106,107 ‘hemical communication, 8-9 ‘hemi equiirum, 60 ‘heme reactions, 60-61, 62-65, ‘chemical equilibrium, oupled rections, 63 hloramphenicol 38 ‘oie shift, 103, hotest, 3 ‘oman, 3,17 ‘omenoie’, 2.3.12, ‘hhymetrypnin, 67,25, ii ci eee, 82,82 ‘overleaf 23 o-opeativity. 70,71 codon 31 oenzyine Q,79 soonayaes, 67 ‘fates, 67 lipase. 7 collagen 39, 40-1 ate of, 40-1 pathophysiology. 41 Symtesis 41 contol elements, 42 Con's disease 83 cotransport. 7 ‘coupled reactions, 63 cst, 3 eateine, 99 ‘ytectrome C79 ytokines, 49 ‘Soseleton, 3 pts 23 deaminaed cytosine repair 19 hydratase. 98 ‘denaturation. 75 tleoxyibose, 12 ‘httsion.2 tigeston, 72-73, sorption of solutes, 73 tarbohydrates, 74.75, Tips, 75-77 pathophysiology. 73 proteins, 74-75 cretion of slates, 73 Ailycerides. 33 ‘imenraton block, 11 Aiploid 2 DNA. repair, /8-19 replication 16,17 tunwinding 28 DNA ligase, 17

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