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Notes in Biochemistry:

Seminar No.1: Proteinogenic Amino Acids

I. Structure and classification of the proteinogenic amino acids

II. Amino acids as ampholytes:
A. dissociation constant of ionogenic groups
B. titration curve, buffer properties
C. isoelectric point

I.
Structure
- carboxyl group
- side chain (“R-group”) bonded to alpha-carbon atom
- at physiological pH (approximately pH 7,4 the carboxyl group is
dissociated forming the negatively charged carboxylate ion (-
COO-), the amino group is protonated (-NH3+)
- peptide linkage
- non polar “even distribution of electrons OR polar (such as
acids and bases)
- Amino acids with non polar side chain do not gain or lose
protons or participate in the hydrogen or ionic bonds
• the side chain of the non polar amino acids tend to cluster
together in the interior of the protein “hydrophobic effect”
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Page 3 of 91
- Prolin’s side chain and alpha-amino N form a rigid,
five-membered ring structure
• “imino acid”
• formation of the fibrous structure of collagen
(often interrupts the alpha-helices found in
globular proteins
- Amino acids with uncharged polar side chains
have zero net charge at neutral pH
• although the side chains of cysteine & tyrosine
can lose a proton at an alkaline pH
• Serine, threonine, and tyrosine contain apolar
hydroxyl group, which can participate in
hydrogen bond formation
• Disused bonds (eg.: cysteine) sulfhydryl group
(-SH)
- the -SH groups of two cysteines can become
oxidised to form a dimer, cysteine , which
contains a covalent cross-link called a
disulfide bond (-S-S-)
• side chains as attachment for other compounds
- polar hydroxyl group of serine, threonine and
rarely tyrosine can serve as a site of
attachment for e.g. a phosphate group
- the amide group of asparagine as well as the
hydroxyl group of serine or threonine can
serve as a site of attachment for
oligosaccharide chains in glycoproteins
- Amino acids with acidic side chains
• aspartic & glutamic acid are proton donors
- >at physiologic pH side chains of lysine
arginine are fully ionised and positively
charged or neutral, depending on the ionic
environment provided by the polypeptide
chains of the protein
Page 4 of 91
- Optical properties of amino acids
• alpha-carbon of an amino acid is attached to four
different chemical groups ->chiral or optical active
carbon atom (exception: glycine)
• amino acids with an asymmetric centre at the
alpha-carbon can exist in two forms, designated D
and L —> stereoisomers, optical isomers, or
enantiomers

II.
- Acidic and basic properties of amino acids
• amino acids in aqueous solution contain weakly acidic alpha-carboxyl groups and weakly basic alpha-amino acid
groups
• acidic and basic amino acids contains an ionisable group in its side chain • can act as a buffer
• acids are proton donor and bases proton acceptors
• weak acids or bases ionise only to a limited extend
• the concentration of protons in aqueous solution is expressed as pH
- pH = log 1/(H+) or -log (H+)
- concentration weak acid (HA) and its conjugated base (A-)
• Derivation of the equation:
dissociation constant of the acid (Ka):

—>the larger the Ka the stronger the acid

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• Buffers:
- resist change in the pH following the addition of an acid or base
- created by mixing a weak acid (HA)with its conjugate base (A-)
- HCl can be neutralised by A-, in this process being converted to HA
- Base can be neutralised by HA, being converted to A-
- maximum buffering capacity occurs at the pH equal to the pKa
• Application of the Handerson-Hasselbach
equation
- dissociation constant of the carboxyl group of
an amino acid called K1 rather than Ka
- used to analyse the dissociation of the carboxyl
group of alanine in the same way as described
for acetic acid:

-when
pH = pK
equal
amounts
of Forms
I & II
- The isoelectric point
(pI)

• Carboxyl groups are ionised, but the net

charge is zero
• the pH at which an amino acid is electrically
neutral, that is , in which the sum of the positive
charges equals the negative charges
- Net charge of amino acids at neutral pH •
negatively charged group (COO-)
• positively charged group (NH3+)
• Glutamate, Aspartate, Histidine, Arginine, and
lysine have additional potentially charged groups
in their side chains
Page 6 of 91
 Page 7 of 91

Page 8 of 91

Seminar No.2: Proteins I Structure

and Functions
I. Peptides - structure and function
II. Protein functions
III. Structure:
A. levels of proteins structure organisation
B. bonds and interactions in protein molecules, role of the
amino acid side chains
IV. Physicochemical properties of proteins
A. denaturation
B. acid-base properties and isoelectric point (pI), buffer
properties
C. solubility and precipitation

- four organisational levels: primary, secondary, tertiary,

quaternary
- increasing complexity
- structural elements range from simple combinations of alpha
helices and beta-sheets forming small motifs, to the complex
folding of polypeptide domains of multifunctional proteins -
Primary structure:
• sequence of amino acids in a protein is called “primary
structure”
• many genetic diseases result in proteins with abnormal amino
sequences, causing improper folding and loss or impairment of
normal function
• amino acids are joined covalently by peptide bonds - amide
linkages between alpha-carboxyl group & the alpha amino
group of another amino acid (e.g.: valine &
alanine dipeptide valylalanine
- not broken by conditions that denature
proteins
(heating or high concentrations of
urea)
- strong acids or bases at elevated
temperatures is required to hydrolyse
these bonds nonenzymically
• free amino end (N-terminal), free carboxyl
end (C terminal)
- sequences are read from N- to
C-terminal end
- each component amino acid residues
have their suffixes changed to -ly (e.g.:
valine, glycine, leucine — >
valylglycylleucine
• characteristics of a peptide bond
- double bond character, shorter than a
single bound, rigid and planar,
preventing free rotation around the
bound
- bonds between the alpha-carbons and
the alpha amino or carboxyl groups can be freely
rotated,
 allowing the polypeptide chain to assume a variety of
possible configurations
- generally a trans bond

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• Polarity of the peptide bond
- -C=O & -NH groups of peptide bond are
uncharged
(neither accept nor release protons over the
pH range
of 2-12
- charged groups: N-terminal (alpha-amino),
C-terminal
(alpha-carboxyl) group
- -C=O & -NH groups are polar and involved in
hydrogen bonds (e.g.: alpha-helices &
beta-sheet)
• Determination of the amino acid composition of
a
polypeptide
- first hydrolysed by strong acid at 110°C for
24h
- separated by cation-exchange
chromatography
- amount of each amino acid is determined
spectrophotometrically by measuring the amount of
light absorbed by the ninhydrin derivative
• Sequencing of the peptide from its N-terminal end
- Phenylisothiocyanate “Edman reagent” used to label
the amino-terminal residue under mildly alkaline
conditions
• Cleavage of the polypeptide into smaller fragments
- enzymes that hydrolyse peptide bonds are termed
peptidases (proteases)
- secondary structure of proteins
• regular arrangements of amino acids that are located near to
each other in the linear sequence
• alpha-helix, beta-sheet, and beta-bend (beta-turn)
• e.g.: collagen alpha-chain helix
• alpha-helix:
- spiral structure consisting of a tightly packed, coiled
polypeptide backbone core
- side chains extend outward from the central axis, avoiding
sterical interference with each other
 - keratins are a family of closely related fibrous proteins whose
structure is nearly entirely alpha-helical
- stabilised by hydrogen bonding between the peptide-bond
carbonyl oxygens and amide hydrogens
• hydrogen bonds extend up and are parallel to the spiral
from the carbonyl oxygen of one peptide bond to the -NH
group of a peptide linkage four residues ahead in the

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polypeptide
- one turn of an alpha helix contains 3.6 amino acids
- Proline disrupts an alpha-helix because its secondary
amino group is not geometrically compatible
- charged amino acids also disrupt the helix by forming
ionic bonds or repelling each other
- amino acids with bulky side chains can interfere with
formation of the alpha-helix if they are present in
large numbers
• beta-sheet
- all of the peptide bond components are involved in
hydrogen bonding
- surfaces appear “pleated” (beta-plated sheets)
- composed of two or more peptide chains (or segments)
arranged parallel or antiparallel
- hydrogen bonds are perpendicular to the
polypeptide
backbone
- when hydrogen bonds are formed
between polypeptide
chains they are termed “interchain
bonds”
- can also be formed by a single
polypeptide chain
folding back on itself > hydrogen bonds
are interchain
bonds
- in globular proteins beta-sheets always
have a right
handed curl or twist
• beta-bend (reverse turn, beta-turns)
- stabilised by hydrogen and ionic bonds
- reverse the direction of polypeptide
chain, helping it
form a compact, globular shape
- found on the surface of protein
molecules and often
include charged residues
- often connecting successive strands of
antiparallel
beta-sheets
 - composed of four amino acids
- proline causes “kink” in polypeptide chain
- glycine often found in beta-bends

• one half of an average globular protein is organised into

repetitive structures —> alpha helix / beta-sheet
• nonrepetitive structures are not random -> simpler less
regular structural elements
• globular proteins are constructed by combing secondary
structural elements forming primarily the core region (the
interior of the molecule) -> connected by loop regions at

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the surface of the protein
• super secondary structures are usually produced by
packing side chains from adjacent secondary structural
elements close to each other
- Tertiary structure of globular proteins
• refers to folding domains
• final arrangement of domains
• structure of globular proteins in aqueous solution is
compact, high-density of the atoms in the core of the
molecule
• hydrophobic side chains are buried in the interior, hydrophilic
groups generally of the surface of the molecule
• Domains
- fundamental functional & three dimensional
structural units - core of a domain is built from
combinations of super
secondary structural elements (motifs)
- folding occurs independently of folding in other
domains
- each domain has the characteristics of a small
compact
globular protein, structurally independent of the
other domains • disulfide bonds:
- covalent linkage formed from sulfhydryl group
(-SH)
- folding rings cysteine residues into proximity,
permits covalent bond of side chains
- contributes to stability of the three-dimensional
 shape
• hydrophobic interactions:
- amino acids with non polar side chains tend to be located in the
interior of the polypeptide molecule, associating with other
hydrophobic amino acids
- segregation of R-groups occurs that is energetically most
favourable
• hydrogen bonds:
- amino acid side chains containing oxygen-, or
nitrogen-bound hydrogen
- can form hydrogen bonds with electron-rich atoms
- enhance the solubility of the protein
• ionic interactions:
- negatively charged groups (COO-) in the side chain of
aspartate or glutamate, can interact with positively
charged groups such as the amino group (NH3+) in the
side chain of lysine
• Protein folding
- interactions between side chains of amino acid
determine how a long polypeptide chain folds into the
intricate three-dimensional shape of a functional
protein
- as a peptide folds the amino acid side chain are
attracted and repulsed according to chemical
properties
- positively and negatively charged side chains attract and
chains with the same charge repel each other
- hydrogen bonds and disulfid bonds exert an influence
on folding - trial and error process mostly
• denaturation of proteins
- unfolding and disorganisation of the proteins secondary
and tertiary structures
• > not accompanied by hydrolysis of peptide bonds
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• heat, organic solvent, mechanical mixing, strong acids or bases, detergents,and
ions of heavy metals (lead, mercury) are denaturing agents
• may be reversible but most proteins remain permanently disordered, often
insoluble, precipitate from solution
• role of chaperons
- information needed for correct protein folding is contained in the primary structure of the
polypeptide
- protein begins to fold in stages during its synthesis
- specialised group of proteins “chaperons” are required
for the proper folding
- Chaperons “heat shock” proteins interact
with the
polypeptide at various stages during the
folding process
- some are important in keeping the
 protein unfolded until
its synthesis is finished or act as catalysts increasing the
rates of the final stages in the folding process, others
protect proteins by folding vulnerable regions, exposed
regions so they do not become tangled in unproductive
interactions
- Quaternary structure
• single polypeptide chain are monomeric proteins
• may consist of two or more chains
• arrangement of these polypeptide subunits is called
Quaternary structure
• subunits are held together by noncovalent interactions
(hydrogen bonds, ionic bonds,hydrophobic interactions)
may either function independently or work cooperatively
- Amyloid disease
• misfiling of proteins may occur spontaneously or be
caused by a mutation in a gene, producing an alert protein
• apparently normal proteins can, after abnormal proteolytic
cleavage, take on a unique conformational state leading to
long fibrillar protein assemblies consisting of beta-pleated
sheets
- accumulation of these insoluble spontaneously
aggregating proteins “amyloids”
- amyloid plaque is amyloid beta in alzheimer containing
40-42 amino acid residues
• deposited in brain and derived from larger amyloid
precursor protein
- aggregated beta-plated-sheet neurotoxic
- Prion disease
• prion protein (PrP) is implicated as the causative agent of
transmissible spongiform encephalopathies (TSEs),
“Creutzfeldt-Jakob disease” in humans, scrapie in sheep
and bovine spongiform encephalopathy in cattle “mad cow
disease”
• this infectious protein is designated PrPsc (Sc=scrapie) is
highly resistant to proteolytic degradation and tends to
form insoluble aggregates of fibrils
• non-infectious is form of PrPc (C=Cellular), present in
normal mammalian brains on the surface of neutrons and
glial cells
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Page 14 91 of

Seminar No.3: Proteins II Globular And Fibrous Proteins 1.

Globular proteins
V. Myoglobin structure and function
VI. Hemoglobin structure and function
A. quaternary structure: subunits and alpha/beta-dimers interactions B. R- and T- forms:
oxygen affinity
VII.Kinetics of myoglobin and hemoglobin oxygen saturation
VIII.Factors affecting Hb affinity oxygen:
A. heme-heme interactions
B. Bohr effect
C. effect of 2,3-bisphosphoglycerate (2,3-BPG)
IX. Methemoglobin and carboxyhemoglobin
X. Minor hemoglobins: HbF, HbA2, HbA1c
XI. Hemoglobinopathies
2. Fibrous proteins
A. collagen: structure features, biosynthesis and degradation
B. elastin: structure features, biosynthesis and degradation

- heme as a tightly bound prosthetic group

- the heme group functions as a electron carrier that is alternately oxidised and reduced
- heme group of the enzyme catalase is part of the active site of the enzyme that
catalyses the breakdown of hydrogen peroxide
- heme group reversible binds oxygen
- heme is a complex of protoporphyrin IX and ferrous iron (Fe^2+)
• the iron is held in the centre of the heme molecule by bonds to the four nitrogens of
the porphyrin ring
• heme Fe^2+ can form two additional bonds, one of these positions coordinate to the
side chain of a histidine residue
• other position is available to bind oxygen
- Myoglobin a hemeprotein present in heart and skeletal muscle is a reservoir for
oxygen and a oxygen carrier
• increases the rate of transport of oxygen within the muscle cell
• single polypeptide chain
• alpha-helical content: 80% of its polypeptide chain folded into eight stretches of alpha
helix
- regions are labeled A-H, terminated either by the presence of proline or by beta bends
and loops stabilised by hydrogen bonds
• interior in composed of non polar amino acids, stabilised by hydrophobic interactions •
charged amino acids are almost entirely on the surface and can form hydrogen bonds •
binding to the heme group:
- one, the proximal histidine (F8), binds to the iron of the heme
- the second or distal histidine does not interact with the heme group • but it helps
stabilising the binding of oxygen to the ferrous iron
 Page 15 91 of
- Hemoglobin is found exclusively in red blood cells, transporting oxygen •
Hemoglobin A:
- 4 polypeptide chains, 2 alpha- & 2 beta chains
- >held together by nonconvalent interactions
- subunits have stretches of alpha-helical structure and a heme binding pocket - can
transport H^+ and CO2 from the tissues to the lungs and 4 O2 molecules back -
regulated by allosteric effectors
- two identical dimers (alpha-beta)1 and (alpha-beta)2
• two polypeptide chains are held within each other dimer tightly, by primary
hydrophobic interactions
• ionic and hydrogen bonds also occur between the members of the dimer, but two
dimers are able to move with respect to each other

- T form: “T”, or taut (tense) Form

• two alpha-beta dimers interact through a network of ionic and hydrogen
bonds, constraining the movement of the polypeptide chains
• low oxygen affinity
- R form: “relaxed” form
• binding oxygen causes rupture of some of the ionic and hydrogen bonds
between the alpha-beta dimers
• polypeptide chains have more freedom of movement
• high oxygen affinity
Page 16 91 of
- myoglobin can bind only one molecule (one heme group) -
hemoglobin can bind 4 oxygen molecules (four heme groups) -
Oxgen dissociation curve:
• myoglobin has a higher oxygen affinity than does
hemoglobin
• Myoglobin (Mb): dissociation curve has a hyperbolic shape •
simple equilibrium: Mb + O2 <—> MbO2
- equilibrium shifts to the right or left depending if oxygen is
added or removed from system
• Hemoglobin (Hb): dissociation curve has a sigmoidal shape -
cooperative binding of four subunits of hemoglobin means that
the binding of an oxygen molecule at one heme
group increases the oxygen affinity of the remaining
heme groups “heme-heme interactions”
- allosteric effects
• ability of hemoglobin to reversibly bind oxygen is
affected by the pO2, pH, partial pressure of carbon
dioxide pCO2,
availability of 2,3-bisphosphoglycerate
- Bohr effect:
• release of oxygen from hemoglobin is enhanced
when the ph is lowered or when pCO2 increases
- >both result in a decreased oxygen affinity, a shift
to the right in the oxygen dissociation curve
• raising the pH and lowering the concentration of
CO2
- >greater affinity for oxygen, shift to the left in the
oxygen dissociation curve and stabilisation of R
state
- >where an increase in protons (or a lower pO2)
shifts the equilibrium to the right, but increase in pO2 (or a
decrease in protons shifts the equilibrium to the left
 -Effect of 2,3-Bisphosphoglycerate on
oxygen affinity:
•important regulator of the binding of oxygen
to hemoglobin
•most abundant organic phosphate in RBC
•2,3-BPG decreases the oxygen affinity of
hemoglobin by binding to deoxyhemoglobin
but not to oxyhemoglobin, stabilising taut
conformation of deoxyhemoglobin
•2,3-BPG binds to pocket by 2 beta-globing
chains positively charged amino acids that
form ionic bonds with negatively charged
phosphate groups of 2,3-BPG
•shifting the oxygen dissociation curve to the
right ->reduced affinity enables release of O2
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• 2,3-BPG in the RBC increases in response to chronic
hypoxia —> in chronic obstructive pulmonary disease
(COPD) like emphysema or high altitudes
• stored blood displays an abnormally high oxygen affinity,
and fails to unload oxygen properly in the tissues
• restore their depleted supplies of 2,3-BPG in 6-24h
- binding of CO2
• CO2 is carried as carbonate bound to the N-terminal amino
groups of hemoglobin —>forming carbaminohemoglobin •
binding of CO2 stabilises the T (taut) or deoxy form, decreases
in its affinity for oxygen and a right shift in the
oxygen dissociation
- binding of CO
• carbon monoxide binds tightly to the
 haemoglobin iron, forming carbon monoxyhemoglobin
• CO binds to 1 or more of the 4 four heme sites
• hemoglobin shifts to the relaxed conformation, causing the
remaining heme sites to bind oxygen with high affinity, shifting
the oxygen dissociation curve to the left and changes the normal
sigmoidal shape toward a hyperbola
• Minor hemoglobins
- Fetal hemoglobin (Hb F)
• 2 alpha-chains identical to those found in Hb A & 2 gamma
chains, gamma chains are from beta-globin gene family • has a
higher affinity for oxygen than does Hb A, weakly binding to
2,3-BPG
- Hemoglobin A2 (Hb A2)
• first appearing shortly before birth (2% of
total
hemoglobin)
• 2 alpha-globin chains and 2 delta-globin
chains
- hemoglobin A1c (Hb A1c)
• under physiologic condition, Hb A in slowly
and
nonenzymatically glycosylated
• glucose residues attached predominantly to
the NH2 groups of the N-terminal valines of
the beta-globin chains • increased amounts of
Hb A1c are found in RBC by
diabetes mellitus
- alpha-gene cluster on chromosome 16 contains 2
genes for the alpha-globoin chains
• > also contains the zeta gene
- beta-gene
• single gene for the beta-globin chain is located
on
chromosome 11
• etha gene (like the zeta gene is expressed early
in embryonic development)
- Hemoglobinopathies
• stickle cell anemia (hemoglobin S disease)
- caused by single nucleotide alteration in the gene for beta
globin
- homozygous recessive disorder
- resulting hemoglobin alpha2-betaS2 is referred to as Hb S -
infant does not show symptoms of disease until sufficient
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Hb F has been replaced by Hb S
- lifelong pain episodes, chronic haemolytic anemia with
associated hyperbilirubinemia
- increased susceptibility to infections
- erythrocyte lifetime is less than 20 days (normal 120 days)
 - glutamate at position six has been replaced with valine (beta
chains
• absence of the negatively charged glutamate residues in the 2
beta-chains
• >rendering Hb S less negative than Hb A
- >the replacement of the charged glutamate with the non polar
valine forms a protrusion on the beta-globin that fits into a
complementary site on the beta-chain of another hemoglobin
molecule in the cell
- >>forming a network of fibrous polymers that stiffen and distort
the cell, producing rigid, misshapen erythrocytes
- sickled cells frequently block the flow of blood in the narrow
capillaries
• localised anoxia
• pain and eventually death (infraction) of cells
• Hemoglobin C disease
- a single amino acid in the sixth position of the beta-globin chain
- > lysine is substituted for glutamate
- relatively mild, chronic haemolytic anaemia
• hemoglobin SC disease
- beta-globin chains have the sickle cell mutation,
whereas other beta-globin chains carry the
mutation found in Hb C disease
- Methemoglobinemias
• oxidation of the heme component of haemoglobin to
the ferric
(Fe^3+) state forms methemoglobin (can not bind
oxygen)
• >oxidation maybe caused by action of certain drugs,
such as
nitrates or endogenous products, also a inherited
defect maybe
possible, furthermore a deficiency of NADH-cytochrome b5
reductase
- mutations in the alpha- or beta-globin chain promote the
formation of methemoglobin
• brownish-blue coloration of the skin and mucous membrane
• tissue hypoxia
- Thalassemias
• imbalance occurs in the synthesis of globin chains
- beta-thalassemias: synthesis of beta-globin is decreased or absent •
>causing premature death
- alpha-thalassemias: synthesis of alpha-globin chains is decreased or absent •
deletional mutations
• if one of the four genes is defective the individual is termed a silent carrier of
alpha-thalassemia
• if two are defective “having a alpha-thalassemia trait”
• three “Hb H (beta4) disease”
- mild to moderate severe hemolytic
 • four alpha-globin genes are defective
- Hb Bart (gamma4) disease —> fetal death because alpha-globin chains are
required for the synthesis of Hb F
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- Collagen and elastin are found as components of skin, connective tissue, blood vessel
walls, and sclera and cornea of the eye
- Collagen is the most abundant protein in the human
body, a rope like triple helix
• more than 25 types
• three polypeptide alpha chains are held together by
hydrogen bonds between the chains
• variations in the amino acid sequence of the alpha
chains result in structural components that are
about the same size but with slightly different
properties
• collagen type I contains 2 chains “alpha1” and 1
chain “alpha2”
• collagen type II contains 3 “alpha1” chains
• >organised in three groups:
- Fibril-forming collagens: Types I, II, III,
 • have characteristic banding patterns, regular stoppered packing of the individual
collagen molecules in the fibril
• type I collagen found in supporting elements of high tensile strength

Page 20 91 of
• type II collagen molecules are restricted to
cartilaginous structures
• type III collagen are prevalent in more distensible
tissues, such as blood vessels
- network forming collagens
• types IV and VII form a 3D mesh
• type IV molecules assemble into a sheet or
meshwork that constitutes a major part of basement
membranes - Fibril-associated collagens: Types IX
and XII bind to the surface of collagen fibrils, linking
these fibrils
• Structure of collagen:
- amino acid sequence:
• rich in proline and glycine
(important for
formation of
triple
helix)—>proline
facilitates the
formation of the
helical
conformation of
each alpha
chain “ring
structure causes
kniks”
•glycine is found
in every third
position of the
polypeptide
chain, fitting into
restricted
spaces
•-Gly-X-Y-,
where X is
frequently
proline and
Y is often
hydroxyproline,
but it can be
hydroxylysine
-triple-helical
structure:
elongated, triple
helical structure
 -hydroxyproline and hydroxylysine
•collagen contains hydroxyproline (hyp) and
hydroxylysine (hyl)
•resulting from the hydroxylation of some of
the proline and lysine residues
-example of post translational modification
•Biosynthesis of collagen
- collagen molecule are formed in fibroblasts
or in related osteoblasts
-secreted into extracellular matrix
-mature collagen monomers aggregate and
become cross linked to form collagen fibers
- Elastin is a connective tissue protein with rubber-like properties •
fibers that are tough and have high tensile strength • it is
insoluble protein polymers synthesised from a precursor,
tropolastin
• rich in proline and lysine BUT only a little hydroxyproline and
hydroxylysine
• tropoelastine is secreted by the cell into the extracellular
space
• interacts with specific glycoprotein microfibrils such as fibrillin •
lysol side chains of the tropoelastin polypeptides are oxidatively
deaminated by lysyl oxidase, forming allysine residues
• alpha1-antitrypsin: inhibits a number of proteolytic enzymes
(also called proteases or proteinases)
- alpha1-AT has the important physiologic role of inhibiting
neutrophil elastase
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 Page 22 91 of

Page 23 91 of

Seminar No.4: Enzymes I

I. Properties the enzymes as biocatalysts
II. Nomenclature and classification of enzyme cofactors
III. Water-soluble vitamins as enzyme cofactors
IV. Isoenzymes - clinical significance
V. Kinetics of the enzyme reaction
A. velocity of the enzymatically catalysed reactions
B. influence of (S), (E), (H+) and t0 on the velocity of the
enzyme reaction

- Nomenclature
• recommended name
- enzyme names have the suffix “-ase”
• systematic name
- are divided into six major classes, each with numerous
subgroups
- suffix “-ase”
- description of the chemical reaction catalysed
- Properties of enzymes: increase the velocity of a chemical
reaction and are not consumed during the reaction
• Active sites contain a special pocket or cleft
- amino acids participate in substrate binding and
catalysis
- Substrate-Enzyme complex (ES)
- ES is converted to Enzyme-Product (EP) complex
• dissociates to enzyme and product
• catalytic efficiency
- number of molecules of substrate converted to product
per enzyme molecule per sec. is called “turnover or Kcat”
• typically 10^2-10^4s^-1
• specificity
- interacting with one or few substrates and catalysing only one type of chemical
reaction
• Holoenzymes: refers to the active enzyme with its nonprotein component - enzyme
without its nonprotein moiety is termed an apoenzyme and is inactive - if the nonprotein
moiety is a metal ion such as Zn^2+ or Fe^3+ it is called a cofactor - if it is a small
organic molecule, it is termed a coenzyme
- if the coenzyme is permanently associated with the enzyme and returned to its
original form, it is called a prosthetic group
• regulation
- enzyme activity can be regulated, that is increased or decreased
• location within the cell
- localised in specific organelles
- serves to isolate the reaction substrate
- How enzymes work
• energy changes occurring during the reaction
- all chemical reactions have an energy barrier separating the reactants and the
products
 • A <—> T<—>B
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- free energy of activation
• because of the high free energy of activation, the rates of
uncatalyzed chemical reaction are often slow
- rate of reaction
• for molecules to react, they must contain sufficient energy to
overcome the energy barrier of the transition state
- alternate reaction pathway
• an Enzyme allows a reaction to proceed rapidly under
conditions prevailing in the cell by providing an alternate
reaction pathway with lower free energy of activation
• the enzyme does not change the equilibrium reaction, but it
accelerates the rate with which equilibrium is reached • chemistry of
the active side
- Transition-state stabilisation
• the active side often acts as a flexible molecular template
that binds the substrate and initiates its converted to the
transition state
• greatly increases the concentration of the
reactive intermediate that can be converted
to a product and, thus accelerates the
reaction
- other mechanisms
• active site can provide catalytic groups that
enhance the probability that the transition
state is formed
- visualisation of the transition state
• high energy of the activation
- Factors affecting the reaction velocity
• different enzymes show different responses to
changes in substrate concentration,
temperature, and pH
• Substrate concentration
- velocity of the reaction (v)
is the
number of substrate
molecules
converted to product per
unit time
• expressed as (mu)mol of
product
formed per minute
• increases with substrate
concentration until a
maximal
velocity (Vmax)
- Michaels-Menten kinetics, in which
the plot of initial reaction velocity (v0)
against substrate concentration (S) is
 hyperbolic
• allosteric enzymes do not follow
Michaelis-Menton
kinetics and
show a sigmoidal
curve
• Temperature
- the reaction velocity
increases with
temperature until a
peak
• > further elevation
of the
temperature
results in a
decrease in
reaction velocity
• >>from temperature induced
denaturation of the enzyme

Page 25 91 of
• pH
- effect of pH on the ionisation of the active site
• the catalytic process usually requires that the
enzymes and
substrate have specific chemical groups in either
an
ionised or unionised state in oder to interact
• extrems of pH can also lead to denaturation
• the optimal pH varies between the enzymes
- Michaels-Menten Equation
• Reaction model:
- enzyme reversibly combines —>ES complex that
subsequently yields

product
•

Michaelis-Menten equation
- describes how reaction
 velocity varies with
substrate conc.

- relative concentrations E
and S
• concentration of substrate (S) is much greater that the
enzyme (E)
• total amount of substrate bound by the enzyme at any time
is small
- steady state assumption
• (ES) does not change with time the rate of formation of ES is equal to that of the
breakdown of ES
• >synthesis is equal to degradation
- initial velocity: (v0)
• is used in the analysis of enzyme reaction
• rate is measured as soon as enzyme and substrate are mixed
• >back reaction from P to S can be ignored
- important conclusions:
• Km the michaelis constant is characteristic of an enzyme and its particular
substrate, and reflects the affinity of the enzyme for that substrate
- a numerically small Km reflects a high affinity of the enzyme for that substrate - a
numerically large Km reflects a low affinity of the enzyme for the substrate • the
relationship of velocity to the enzyme concentration: the rate of the reaction is to the
enzyme concentration at all concentrations
- order of reaction
• when S is much less than Km the velocity of the reaction is approximately
proportional to the substrate concentration
• when S is much greater than Km the velocity is constant and equal to Vmax -
>the rate of reaction is then independent of substrate concentration
Page 26 91 of
• Lineweaver-Burk plot
- v0 is plotted against S it is
not always possible to determine when Vmax is achieved
- > if 1/v0 is plotted versus 1/S, a straight line is obtained
- can be used to calculate Km and Vmax
Page 27 91 of

Seminar No.5: Enzymes II Regulation Of Enzyme Activity

I. Inhibition of enzyme active:

 A. irreversible inhibition
B. reversible inhibition - competitive and noncompetitive inhibition
II. Regulation of enzyme activity by affecting their synthesis and degradation - constitutive
inducible enzymes
III. Allosteric enzmes
A. properties
B. allosteric efforts (modulators)
C. kinetics of allosteric enzymes, cooperative effect
D. regulation of allosteric enzymes activity
IV. Reversible covalent modification
V. Limited proteolysis

- Any substrate that can diminish the velocity of an enzyme-catalysed reaction is called an
inhibitor
- irreversible inhibitor bind to enzymes through covalent bonds
- reversible inhibitors bind to enzymes through nonconvalent bonds
- competitive inhibition occurs when the inhibitor binds reversibly
• Effect on Vmax:
- increasing concentration of (S) to achieve Vmax
• Effect on Km:
- a competitive inhibitor increases the apparent Km for a given substrate
- more substrate is needed to achieve 1/2 Vmax
• effect on the Lineweaver-Bruck plot:
- plots of the inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax -
different x-axis intercept
- Km is increased in the presence of the competitive inhibitor

Page 28 91 of
- nocompetetive inhibition has an effect of Vmax
• inhibitors occurs when the inhibitor and substrate bind at different sites on the
enzyme
• Effect on Vmax
- inhibition can not be overcome by increasing
substrate
concentration
- decreasing the Vmax
• Effect on Km
- do not interfere with the binding of substrate to
enzyme —>
same Km
• Effect on Lineweaver-Burk plot:
- Vmax decreases
- Km is unchanged
• Example:
- lead forms a covalent bond with the sulfhydryl
side chain of
cysteine in protein
• heavy metal shows noncompetitive inhibition
- penecillin and amoxicillin act by inhibiting enzymes involved in
bacterial cell wall synthesis
- Regulation of enzyme activity
• regulation of allosteric enzymes
- regulated by molecules called effectors (also “modifiers”)
- binding noncovalently at a site other than the active site
• enzymes are usually made up of multiple subunits
- when the substrate itself serves as an effector, the effect is said to
be homotropic
• an allosteric substrate functions as a positive effector
• the presents of a substrate molecule at one site on the enzyme
enhances the catalytic properties of the other substrate-binding
sites , that is their binding sites exhibit cooperatively
- these enzymes show a sigmoidal curve when reaction velocity
(V0) is plotted against substrate concentration
- effector might by different from the substrate, in which case the

Page 29 91 of
effect is said to be heterotopic
• feedback inhibition
• the enzyme that converts D-E has an allosteric
site that
binds to end product G
- G increases first irreversible step unique to the
pathway is typically inhibited
• regulation of enzymes by covalent modification
- Many enzymes may be regulated by covalent
modification
• addition or removal of phosphate groups from
specific
serine, threonine, or tyrosine residues of the
enzyme
- phosphorylations are catalysed by a family of
enzymes
called protein kinases that use adenosine
triphosphate
 (ATP) as a phosphate donor
- the phosphorylated form may be more or less active than
the unphosphorylated enzyme
• induction and repression of enzyme synthesis
- cells can also regulate the amount of enzyme present by
altering the rate of enzyme degradation or more typically
the rate of enzyme synthesis
- enzymes that are in constant use are usually not regulated
by altering the rate of enzyme synthesis
• alteration in enzyme levels as a result of introduction or repression of protein
synthesis are slow

- Enzymes in clinical diagnosis

• Plasma enzymes can be classified into two major groups
- 1. small group of enzymes are actively secreted into the blood
- 2. a large number of enzymes species are released from cells during normal cell
turnover -> almost always function intracellularly, no physiological use in the plasma •
alteration of plasma enzyme levels in disease states
- diseases that cause tissue damage result in an increased release of intracellular
enzymes into the plasma (diseases of the heart, liver, skeletal muscle and other
tissues)
- alanine aminotransferase (ALT) in abundant in the liver —>elevated levels signal
possible damage to hepatic tissue
- plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of
myocardial infarction
- creatine kinase occurs in three isoenzymes each is composed of two polypeptide
(called B and M subunits) CK1=BB, CK2=MB, CK3=MM
Page 30 91 of
• in the brain BB is the isoform
• in skeletal muscle MM
• in cardiac muscle 1/3 is MB the rest is MM
- diagnosis of myocardial infarction (MI)
• isoenzymes appears approximately 4-8h
following the onset of chest pain
• peak of activity after around 24h
• returns to baseline after 48-72h
• Troponin T and Troponin I are regulatory
 proteins involved in myocardial contractility
• cardiac troponin I (cTnI) specific for heart damage -
appears in plasma within 4-6h
- after MI
- peaks in 8-28h and remains elevated for 3-10
days
-
Page 31 91 of

Seminar No.6: Colloquium On Aminoacids, Proteins And

Enzymes Experimental Work On Enzymes

1.Proving the absolute specificity of urease Principle:

Urease catalyses the hydrolysis of urea: The ammonia product can be visualized by
phenolphthalein (pink color). Urease is a highly specific enzyme and cannot catalyse the
hydrolysis of compound with very similar structure like biuret (H2N-CO-NH2-CO NH2) and
thiourea (H2N-CS-NH2).
Procedure:
In three test tubes pipette 1 ml solution of urease. In separate tubes add 5 ml 0,1M solution
of urea, biuret and thiourea respectively. Add 3-4 drops of phenolphthalein. Incubate for 20
min at room temperature. In the test tubes where the substrate is hydrolyzed a pink color
develops.

H2N-CO-NH2+ H2O ——>CO2+ 2 NH3

2.Calculation of Km and Vmax by Lineweaver-Burke method:

The following data are from kinetic measurements of enzymatic reactions in the presence
and
absence of inhibitor. V′ and V′′ denote the initial speed for the uninhibited and the
inhibited reaction respectively
at different substrate concentrations
Page 32 91 of

Seminar No.7: Bioenergetics I

I. Biological oxidation. Macroergic compounds

A. oxidation-reduction processes
B. redox potential, link between deltaG’° and deltaE’°
C. ATP cycle and role of ATP in the cellular bioenergetics and metabolism II. Oxidative
phosporylation
A. substrate phosphorylation
B. respiratory phosphorylation
III. Structure of the respiratory chain: complex comprising the
respiratory chain and transport of electrons and protons in the
respiratory chain
IV. Coupling of respiratory and phosphorylation - Mitchel’s
chemiosmotic theory. Inhibition of electron transport
V. Fate of cytosolic NADH - shuttles for transport of reducing
equivalents

- Bioenergetics discribes the transfer and utilisation of energy in biologic

 systems (“biochemical thermodynamics”)
- Free energy:
• direction and extend to which a chemical reaction proceeds is determined by 2 factors
- enthalpy (deltaH, a measure of the change in heat content of the reactions and
products)
- entropy (deltaS, a measure of the change in randomness or
disorder of reactants and products
- > when combined mathematically they define a third quantity=> free energy G
• predicts the direction in which a reaction will spontaneously
proceed
- Free energy change
• change in free energy is represented in 2 ways deltaG and deltaG° - deltaG the
change in free energy
- >direction of a reaction at any specific concentration of products and reactions and
products are at a concentration of 1 mol/L
• predict the direction of a reaction
A<—>B
- a negative number means a net loss of energy
• reaction goes spontaneously (A is converted to B) “exergonic” - a positive number is
net gain of energy
• does not go spontaneously from B to A
- >energy must be added “endergonic”
- if deltaG is zero the reactions are in an equilibrium
• free energy is being lost
• energy of the forward reaction (A—>B) is equal magnitude but opposite in sign to that
of the back reaction (B—>A)
• deltaG depends on the concentration of reactions and products

Page 33 91 of
• the standard free energy change, deltaG°, is so called because it is
equal to the free energy change, reactants and products are at 1
mol/L concentration
- under standard conditions, deltaG° can be used to predict the
direction a reaction proceeds, because deltaG° is equal to deltaG •
deltaG° cannot predict the direction of a reaction under
physiologic conditions
- in a reaction A—>B, point of equilibrium is reached at which no
further net change takes place
- >concentration of
the two
compounds :
• allowed to go to
equilibrium at
 constant temperature and
pressure
• at equilibrium the overall free
energy change (deltaG) is
zero:

- ATP as an energy carrier

• processes that have a large positive deltaG
• are made possible by coupling the endergonic
movement of ions with a second, spontaneous
 process with a large negative deltaG
- > exergonic hydrolysis of adenosine triphosphate
(ATP)
• Reactions are coupled through common
intermediates:

Page 34 91 of
- Electron transport chain:
• intermediates of these reactions donate electrons to specific
coenzymes—nicotinamide adenine dinucleotide (NAD^+) and
flavin adenine dinucleotide (FAD)—to form the energy-rich reduced
coenzymes, NADH and FADH2
- > donate a pair of electrons
• energy can be captured and stored by the production of ATP form
ADP and inorganic phosphate —>This process id called oxidative
phosphorylation
- reminder of free energy is used to drive ancillary reactions such
as Ca^2+ transport into mitochondria and to generate heat
• Mitochondrion
- electron transport chain is present in the inner mitochondrial
membrane and is the final common pathway by which electrons
derived from different fuels of the body flow to oxygen
- membranes:
• components of the electron transport chain are located in the
inner membrane
• outer membranes contains special pores, permeable to most
ions and small molecules
• inner membrane impermeable to small ions (H^+, Na^+, K^+,
and small molecules ATP, ADP, Pyruvate)
- specialised carries or transport systems are required
- rich in protein
• Organisation of the electron chain:
- five separate protein complexes I, II, III, IV, V
- each complex accepts or donates electrons to relatively mobile
electron carriers such as coenzyme Q & cytochrome c
• ultimately combine with oxygen and protons to form water
• Complex V is a protein complex that contains a domain (F0)
that spans the inner mitochondrial membrane and a domain
 (F1) that appears as a sphere that protrudes into the
mitochondrial matrix
• Complex catalyses ATP synthesis “ATP synthatase”
• reactions of the electron transport chain
- with the exception of coenzyme Q, all members of this chain are
proteins
• may function as enzymes
Page 35 91 of
• dehydrogenase may contain iron as part of an iron-sulfur center, may be
coordinated with a porphyrin ring as in the cytochromes, or may contain copper as
does the cytochrome a + a3 complex
- Formation of NADH: NAD^+ is reduced to NADH by
dehydrogenase that remove 2 hydrogen
atoms
• both electrons but only one proton are
transferred to the
NAD^+ forming NADH plus a free
proton, H^+
- NADH dehydrogenase: the free proton
plus the hydride ion
carried by NADH are next transferred to
NADH
dehydrogenase (Complex I)
• Complex I has a tightly bound
molecule of flavin
mononucleotide
- >FMN a coenzyme structurally
related to FAD
- accepts two hydrogen atoms —> FMNH2
- NADH dehydrogenase also contains iron atoms paired
with sulphur atoms to make iron-sulfur centers
• necessary for the transfer of the hydrogen atoms to
the next member of the chain, coenzyme Q
- Coenzyme Q: is a quinone derivative with a long,
hydrophobic iosprenoid tail
• mobile carrier, can accept hydrogen atoms both form FMNH2, produced on
NADH dehydrogenase (Complex I) and form FADH2,
produced on succinate
dehydrogenase (Complex II),
glycerophosphate dehydrogenase,
and acyl CoA
dehydrogenase
• CoQ transfers electrons to Complex
III
• CoQ, then, links the flavoproteins to
the cytochromes
- Cytochromes
• the remaining members of the
electron transport chain are
cytochromes -> containing a heme
group
 - >reversibly converted from its freeic (Fe^3+ to Fe^2+)
- reversible carrier of electrons
• electrons are passed along the chain from CoQ to
cytochromes bc1 (complex III), c, and a+a3 (complex IV)
- Cytochrome a+a3
• only electron carrier in which the heme iron has an
available coordination site that can react directly with O2
- >cytochrome oxidase
• transported electrons, O2, and free protons are brought
together, O2, and free protons are brought together, and
O2 is reduced to water
• cytochrome oxidase contains copper atoms that are
required for this complex reaction to occurs
- Site-specific inhibitors
• prevent a passage of electrons by binding to a component
of the chain, blocking the oxidation/reduction reaction
• all electron carries are fully reduced, after the block are
oxidised —>inhibits ATP synthesis
- Release of free energy during electron transport
• free energy is released as electrons are transferred along
the electron transport chain from electron donor to an
electron acceptor
Page 36 91 of
- can be transferred as hydride ions (:H^-) to NAD^+, as hydrogen atoms (H) to FMN,
coenzyme Q, and FAD, or as electrons (e^-) to cytochromes - Redox pairs: oxidation
(loss of electrons) one
compound is always accompanied by reduction (gain of
electrons) of a second substance
• oxidation of NADH to NAD^+ accompanied by the
reduction of FMN to FMNH2
• redox pairs differ in their tendency to lose electrons
- Standard reduction potential (E0)
• the more negative the E0 of a redox pair
- the greater the tendency of the reductant member
of that pair to lose electrons
- the more positive the E0 the greater the tendency
of the oxidant member of that pair to accept
electrons
- DeltaG° in related to deltaE0: the change in the free
energy is related directly to the magnitude of the
change in E0
 - Delta of ATP
• ADP to ATP is +7,3 kcal/mol
• the transport of a pair of electrons from
NADH to oxygen via the electron transport
of a pair of electrons from NADH to oxygen
via the electron transport chain produces
52,58 kcal

Page 37 91 of

- Oxidative Phosphorylation: NADH is a strong electron

donor and molecular oxygen is an avid electron
acceptor
• Chemiosmotic hypothesis: how the free energy
generated by the transport of electrons by the
electron transport chain is used to produce ATP from
ADP + Pi
- Proton pump: transport “pumping” of protons (H^+)
across the inner mitochondrial membrane at
Complexes I, III, and IV • creating an electrical and pH gradient,
sufficient to drive ATP synthesis
 - ATP synthase: enzyme complex ATP synthase (Complex V) •
synthesised ATP using the energy of the proton gradient
generated by the electron transport chain
• after protons are pumped, they reenter the matrix by
passing through a channel in the membrane-spanning
domain (F0) of complex V, driving rotation of F0, and
dissipating the pH and electrical gradients
- F0 rotation causes conformational changes in the extra
membranous F1 domain that allow it to bind ADP+Pi,
phosphorylate ADP to ATP, release ATP
• Oligomycin: This drug binds to the F0 domain of ATP
synthase, closing the H^+ channel, preventing reenters of
protons into the mitochondrial matrix ->preventing
phosphorylation of ADP to ATP
- >electron transport stops because of the difficulty of
pumping any more protons against the steep gradients -
>>dependency of cellular respiration on the ability to
phosphorylate ADP to ATP is known as respiratory
control
- >>>inhibition of one process inhibits the other
• Uncoupling proteins (UCP) occur in the inner
mitochondrial membrane
- create a “proton leak”, allowing protons to re-enter
mitochondrial matrix without energy being captured
- energy is released in heat “nonshivering thermogenesis” •
UCP1 “thermogenin” —> heat production in the brown
adipocytes
- activated by fatty acids —>brown fat uses almost
90% of its respiratory energy for thermogenesis in
response to cold in the neonate, and during arousal in
hibernating animals
• Synthetic uncouplers:
- increase the permeability of inner mitochondrial
membrane to protons
- E.g.:2,4-dinitrophenol, lipophilic proton carrier, diffusing
through mitochondrial membrane, causing electron
transport to proceed at a rapid without establishing a
proton gradient—>energy is released in heat
• aspirin & other salicylate uncouple oxidative
phosphorylation ->toxic overdose is accompanied by
fever
Page 38 91 of
• Membrane transport systems: inner mitochondrial membrane is impermeable to most
charged or hydrophilic substances; contains numerous transport proteins, permitting
passage of specific molecules from cytosol
- ATP-ADP transport: inner mitochondrial membrane requires specialised carriers to
transport ADP and Pi from the cytosol
• where ATP can be resynthesized
• >an adenine nucleotide carrier imports one molecule ADP, exporting one ATP -
Transport of reducing equivalents: inner membrane lacks an NADH transporter, and
 NADH produced in the cytosol cannot directly enter the mitochondrial matrix • >using
shuttles
• >>glycerophosphate shuttle, two electrons are transferred from NADH to
dihydroxyacetone phosphate by cytosolic glycerophosphate dehydrogenase • glycerol
3-phosphate produced is oxidised by the mitochondrial isozymes as FAD is reduced to
FADH2
• CoQ oxidises FADH2
- results in the synthesis of two ATPs for each cytosolic NADH oxidised by
malate dehydrogenase as oxaloacetate is reduced to malate
• defects in oxidative phosphorylation are more like a result of alterations in mtDNA,
which has a mutation rate about ten times greater than that of nuclear DNA
Page 39 91 of
Page 40 91 of

Seminar No.8: Bioenergetics II Shortened Electron

Transport Chains. Free-Radical Oxidation And Antioxidant
 Defense

I. Extramitochondrial oxidation: Shortened electron-transport chains - organisation and

role
A. Desaturation system
B. Cytochrome P450-shortened electron transport chains
II. Free radical oxidation
A. Reactive Oxygen Species (ROS) - types and sources in the live organism B. Role of
ROS
C. Antioxidant defence:
1. intracellular antioxidant defence
2. extracellular antioxidant defence

- Oxygenases are concerned with the synthesis or degradation of many different types
of metabolites
• catalysing the incorporation of oxygen into substrate molecule in two steps - 1.
oxygen is bound to the enzyme at the active site
- 2. bound oxygen is reduced or transferred to the substrate
• two subgroups
- dioxygenases incorporate both atoms of molecule Oxygen into the substrate •
A+O2—>AO2
- monooxygenases (mixed-Function Oxidases, Hydrolases) incorporate only one atom
of the molecular oxygen into the substrate

Page 41 91 of
 • A—H+O2+ZH2—>A—OH+H2O+Z
• Cytochrome P450 are Monooxygenases Important in steroid metabolism & for the
detoxification of many drugs
- located mainly in the in the endoplasmic reticulum in the liver and intestine,
also found in mitochondria in some tissues

- Free Radicals are formed in the body under normal conditions, causing damage to
nucleic acids, proteins and lipids in cell membranes and plasma lipoproteins • can
lead to cancer, atherosclerosis and coronary artery disease
• protective antioxidant nutrients selenium, vitamins C and E, beta-carotene, and other
carotenoids, and a variety of polyphenolic compounds derived from plant foods
Page 42 91 of

Seminar No.9: Metabolism of Carbohydrates I

1. Digestion of carbohydrates
C. basic dietary carbohydrates
D. enzymes responsible for the digestion of carbohydrates
E. absorption of carbohydrates - disturbances in carbohydrates absorption
2. Glycolysis
A. mechanism and biological significance
B. hexokinases - role of different isoenzymes
C. key enzymes and their regulation
D. anaerobic glycolysis - special features and significance
3. Experimental work: Proving of alcohol fermentation products

1. A.
- Empiric formular of simpler carbohydrates
(CH2O)n
- Carbohydrates that have a free carbonyl
group have the suffix -ose
- Monosaccharides can be linked by
glycosidic bonds to create larger structures
• disaccharides contain two,
oligosaccharides three to ten
monosaccharides
- compounds that have the same chemical
formulas but different structures are
isomers
- structures that are mirror images of each
other are enantiomers = D- & L-
• majority of sugars in human body are D-sugars

- 1% of monosaccharides with 5 or more carbons exist in open

chain form
• predominantly in ring form
 - if the hydroxyl group on the anomeric carbon of a cycled sugar is
not linked to another compound by a glycosidic bond, the ring
can open
• > the sugar can act as a reducing agent termed a reducing
sugar
- Important disaccharides:
• lactose (galactose + glucose), sucrose (glucose + fructose),
maltose (glucose + fructose), Glycogen (from animal), starch
(plant sources)
• alpha-bond or beta-bond

Page 43 91 of
- N- and O-glycosides:
• if the non-carbonate molecule to which the sugar is
attached is an - NH2 group: N-glycoside / OH-group is
O-glycoside
B.
- digestion in mouth and intestinal lumen
- digestion is rapid catalysed by enzymes known as glycoside
hydrolases (glycosidases) —>hydrolysing glycosidic bonds •
enzymes are primarily endoglycosidases that hydrolyse
di-/poly-/ oligosaccharides
• di-/trisaccharides are hydrolysed into their reducing sugar
components
- final products of digestion are monosaccharides, glucose,
galactose and fructose
- in mouth alpha-amylase acts briefly on dietary starch •
>hydrolysing random alpha(1-4) bonds
• >>humans do not produce beta(1-4)-endoglucosidases
hence they can not digest cellulose
• branched amylopectin & glycogen also contain alpha(1-6)
bonds — > alpha-amylase can not hydrolyse
- acidic stomach contents reaches small intestine, is
neutralised by bicarbonate from pancreas
• alpha-amylase continues the process of starch digestion -
finally digestion by enzymes synthesised by the upper
jejunum • isomaltase cleaves the alpha(1-6) in isomaltose
• maltase cleaves maltose maltotriose each producing
glucose and fructose
• lactase (beta-galactosidase) cleaves lactose producing
galactose and glucose
• Trehalose, an disaccharide of glucose is cleaved by
Trehalase
C.
- duodenum and upper jajunum absorb the bulk
of the dietary - glucose and galactose are
transported by an energy needing process that
requires current uptake of sodium ions
• transportprotein is sodium-dependent glucose
cotransporter 1 (SGLT-1)
- fructose uptake requieres a sodium-independent
monosaccharides transporter (GLUT-5)
- all three monosaccharides are transported from the intestinal
mucosal cell into the portal circulation by jet another
transporter GLUT-2
- any defect in a specific disaccharide activity of the intestinal
mucosa causes the passage of undigested carbohydrates into
the large intestine
• >water is drawn from the mucosa into the large intestine
causing osmotic diarrhoea
- disaccharide intolerance can stem from: genetics, intestinal
disease, malnutrition, drugs that injure the mucosa of the
small intestine
- lactose intolerance:
• 90% of african and asian decent
• less able to metabolise lactose
Page 44 91 of
• age-dependent loss of lactase activity
• small variation in the DNA sequence of a region on chromosome 2
- sucrase-isomaltase complex deficiency:
• intolerance of ingested sucrose
• 10% inuit people and 2% of north americans are heterozygous for the deficiency
Page 45 91 of
2. A.
- In a pathway the product of one reaction serves as the substrate of the subsequent
reaction
• either catabolic (degenerative) or anabolic (synthetic)
- catabolic reactions serve to capture chemical energy in the form of adenosine
triphosphate (ATP) from the degradation of energy rich fuel molecules
- molecules are converted into building blocks needed for the
synthesis of complex molecules
Page 46 91 of
- energy generation by degradation of complex molecules occurs in three stages
- some energy is captured as ATP in stage II
- The Tricarboxylic acid (TCA) Cycle is the final common pathway
in the oxidation of fuel molecules that produce acetyl CoA
- oxidation of acetyl CoA generates large amounts of ATP via
oxidative phosphorylation as electron flow from NADH and
FADH2 to oxygen
- anabolic reactions combine small molecules, such as proteins
• require energy (are endergonic)
• involve chemical reaction in which the reducing power is most
frequently provided by the electron donor NADPH
• catabolism is a convergent process;
- wide variety of molecules are transformed into a few
common end products
• anabolism is a few biosynthetic precursors from a wide variety
of polymeric or complex products
- Regulation of metabolism
• signals from within the cell (intracellular)
- influence the availability of substrates, product inhibition, or
alterations in the levels of allosteric activators or inhibitors
 - “moment-to-moment” regulation
• communication between cells (intercellular)
- long-range integration of metabolism
- response is slower than is seen with signals that originate
within the cell
- by cell-to-cell contact and formation of gap junctions
- most important route of communication is chemical singling
between blood borne hormones or by neurotransmitters
• second messenger system
- intervene between the original messenger (the
neurotransmitter or hormone and the ultimate effect on cell
- the calcium/phosphatidylinositol system and the adenylyl
system
• Adenylyl cyclase
- membrane receptors
• beta— & alpha2—adrenergic receptors
• >increase or decrease activity of adenylyl cyclase

Page 47 91 of
• a membrane bound enzyme that converts ATP to
3’,5’- adenosine monophosphate “cyclic AMP” or
“cAMP”
• G protein-coupled receptors (GPCR)
- characterised by extracellular ligand-binding
receptors, seven transmembrane helices, and an
intracellular domain that interacts with G proteins
- GTP-dependent regulatory proteins
• mediated by specialised trimeric proteins (alpha, beta,
gamma subunits)
• referred to as G proteins because they bind
guanosine nucleotides form a link in the chain of
communication between the receptor and adenylyl
cyclase
- in the inactive form of a G protein the alpha-subunit
is bound to GDP
- binding of ligand causes a conformational change in
the receptor, triggering replacement of this GDP with
GTP
- the GTP-bound form the alpha subunit dissociates
from the beta-gamma subunits and moves to
adenylyl cyclase
- the actions of the Galpha-GTP complex are
short-lived because Galpha has an inherent GtPase
activity,
- resulting in the rapid hydrolysis of GTP to GDP
- Protein kinases
• activation of e.g.: Protein kinase A by cAMP binding to
its two regulatory subunits, causing the release of
 active catalytic subunits
• the active subunits catalyse the transfer of phosphate from
ATP to specific serine or threonine residues of protein
substrates
- Dephosporylation of proteins
• the phosphate groups added to protein kinases are removed by
protein phosphatases
- Hydrolysis of cAMP
• cAMP is rapidly hydrolysed to 5’-AMP by cAMP
phosphodiesterase
• 5’-AMP is not an intracellular signaling molecule
- The transport of glucose into cells

Page 48 91 of
• two mechanisms: a Na^+independent, facilitated diffusion
transport system or a Na^+ monosaccharide cotransporter
system - designated GLUT-1 to GLUT-14
- two conformational states
- extracellular glucose binds to the transporter , which then
alters its conformation, transporting glucose across the
cell membrane • tissue specificity of GLUT gene
expression
- e.g. GLUT-3 is the primary glucose transporter in
neutrons - GLUT-1 is abundant in erythrocytes and blood
barrier - GLUT-4 is abundant in adipose tissue and
skeletal muscle • specialised functions of GLUT isoforms
- glucose movement follows the concentration gradient -
GLUT-1,-3,-4 are primally involved in glucose uptake from the
blood
- GLUT-2 is found in liver and kidney can either transport
glucose into these cells when blood glucose levels
are low • Na^+ monosaccharide cotransporter
system
- energy-requiring process that transport glucose
“against” a concentration gradient
- system is carrier mediated
- coupled to the concentration gradient of Na^+,
which is transported into the cell into the cell
at the same time—->
sodium-dependent-glucose transporter or
SGLT
- this type of transport occurs in the epithelial
cells of the intestine, renal tubules, and
choroid plexus
B. + C.
- Reaction of glycolysis
- Phosphorylation of glucose
• Phosphorylated sugar molecules do not readily penetrate the cell
 membrane, no specific transmembrane carriers and too polar to
diffuse lipid core of membranes
• phosphorylation of glucose is irreversible
• sugar is trapped as cytosolic glucose 6-phosphate
- Hexokinase:
• phosphorylation of glucose is catalysed by hexokinase •
hexokinase has brod substrate specificity
• is inhibited by the reaction product glucose 6-phosphate •
low Km, so high affinity for glucose
• has low Vmax for glucose cannot sequester (trap) cellular
phosphate in the form of phosphorylate hexoses
- Glucokinase:
• in liver parenchymal cells
• beta cells of the pancreas
• glucokinase (hexokinase D or type
IV) is the
predominant enzyme responsible for
the
phosphorylation of glucose
• functions as a glucose sensor in beta
cells,
determining the threshold for insulin
secretion
• facilitates glucose phosphorylation
during
hyperglycemia
• Kinetics:
- much higher Km than hexokinase

Page 49 91 of
- higher glucose saturation for half-saturation
- glucokinase functions only when when the intracellular
concentration of glucose in the hepatocyte is elevated
- glucokinase has a high Vmax
• allowing the liver to effectively remove the flood of
glucose
delivered to the portal blood
• Regulation by fructose 6-phosphate and glucose
- is inhibited by fructose 6-phosphate and indirectly stimulated by
glucose
- Glucokinase regulatory protein (GKRP) in the liver regulates the
activity of glucokinase through reversible binding
- in presence of fructose 6-phosphate glucokinase
is translocated into the nucleus and binds tightly to
the regulatory protein,
rendering the enzyme inactive
- when glucose levels in the blood increase,
glycokinase is
released from the regulatory protein, enzyme
 reenters the
cytosine where it phosphorylates glucose to glucose 6-
phosphate
- Isomerization of glucose 6-phosphate
• isomerization of glucose 6-phosphate to fructose 6-phosphate is
catalysed by phosphoglucose isomerase
- Phosphorylation of fructose 6-phosphate
• catalysed by phosphofructokinase-1 (PFK1)
• controlled by the availableroncentrations of the substrates ATP
fructose 6-phosphate
• Regulation by energy levels within the cell:
- PFK-1 is inhibited allosterically by elevated levels of ATP,
elevated levels of citrate inhibit PFK-1, conversely PFK-1 is
activated allosterically by high concentrations of AMP, which
signal that the cells energy stores are depleted
• Regulation by fructose 2,6-bisphosphate: —most potent activator of PFK-1 - formed
by phosphofructokinase-2 (bifunctional protein kinase activity “producing fructose
2.6-bisphosphate” and phosphatase activity “dephosphorylates 2,6- bisphosphate
back to fructose 6-phosphate”)
• during the well fed state: —> decreased levels of glucagon & elevated levels of
insulin, after carbohydrate-rich meal, cause an increase in fructose 2,6-
bisphosphate, thus in the rate of glycolysis
Page 50 91 of
• during starvation: elevated levels of glucagon and low
levels of insulin, decrease the intracellular
concentration of hepatic fructose 2,6-bisphosphate,
this result decreases overall the rate of glycolysis
- Aldolase cleaves fructose 1,6-Phosphate to
dihydroxyacetone phosphate and glyceraldehyde
3-phosphate (—>reversible and not regulated)
- Isomerization of dihydroxyacetone phosphate
• triode phosphate isomerase interconverts dihdoxyacetone
phosphate and glyceraldehyde 3-phosphate
• dihydroxyacetone phosphate must be isomerized to
glyeraldehyde 3-phosphate for further metabolism by the
glycolytic pathway • two molecules of glyceraldehyde
3-phosphate from the cleavage products of fructose
1,6-bishosphate
- Oxidation of glyceraldehyde 3-phosphate
• glyceraldehyde 3-phosphate to 1,3-bisphosphoglyerate by
glyceraldehyde 3-phosphate dehydrogenase
- first step in oxidation-reduction reaction of glycolysis - only limited
NAD^+ is present so NADH formed by this reaction of glycolysis
must be deoxidised to NAD^+:
• 1) NADH-linked conversion of pyruvate to lactate
• 2) oxidation of NADH via the respiratory chain
• synthesis of 1,3-bisphosphoglycerate (1,3-BPG)
- oxidation of the aldehyde group of glyceraldehyde 3-phosphate to
a carboxyl group is coupled to the attachment of Pi to the carboxyl
 group
- mechanism of arsenic poisoning
• arsenic toxicity is primarily explained by the inhibition of
enzymes such as pyruvate dehydrogenase, requiring lipoic
acid as a coenzyme
• pentavalent arsenic (arsenate) prevents net ATP & NADH
production by glycolysis without inhibiting the pathway itself -
poison is competing with inorganic phosphate as a substrate
for glyceraldehyde 3-phosphate dehydrogenase, forming a
complex that spontaneously hydrolyses to form 3-
phosphoglycerate —->cell is deprived of energy from
glycolytic pathway
• synthesis of 2,3-bisphosphoglycerate(2,3-BPG) in RBC -
1,3-BPG in converted to 2,3-BPG by the action of
bisphosphoglycerate mutase
- 2,3-BPG is hydrolysed by a phosphatase to 3-
phosphoglycerate
- synthesis of 3-phosphoglycerate producing ATP
• When 1,3-BPG is converted to 3-phosphoglycerate, the high
energy phosphate group of 1,3-BPG is used to synthesise ATP
from ADP
- catalysed by phosphoglycerate kinase (physiologically
reversible)
- because two molecules of 1,3-BPG are formed from each
glucose molecule, this kinase reaction replaces the two ATP
molecules consumed by the earlier formation of glucose 6-
phosphate and fructose 1,6-bisphosphate
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- Shift of the phosphate group from carbon 3 to carbon 2
• phosphoglycerate mutase is freely reversible
- Dehydration of 2-phosphoglycerate
• dehydration of 2-phosphoglycerate by enolase redistributes the energy within the
2-phosphoglycerate molecule
- resulting in formation of phosphorenolpyruvate (PEP)
- Formation of pyruvate producing ATP
• conversion of PEP to pyruvate is catalysed by pyruvate kinase, third irreversible
reaction of glycolysis
• feed-forward regulation:
- pyruvate kinase is activated by fructose 1,6-bisphosphate, the product of the
phosphofructokinase reaction
- linking the two kinase reactions:
• increased phosphofructokinase activity
results in elevated
levels of fructose 1,6-bisposphate, which
activates pyruvate
kinase
• covalent modulation of pyruvate kinase
- phosphorylation by a cAMP-dependent
protein kinase leads
to inactivation of pyruvate kinase in liver
 - when blood glucose levels are low, elevated glucagon
increases the intracellular level of cAMP, which causes the
phosphorylation and inactivation of pyruvate kinase
- PEP is unable to continue in glycolysis, but instead enters the
glyconeogenesis pathway observed inhibition of hepatic
glycolysis and stimulation of glyconeogenesis by glucagon
- dephosphorylation of pyruvate kinase by a phosphoprotein
phosphatase results in reactivation of the enzyme
• pyruvate kinase difficency
- erythrocytes lack mitochondria, completely dependent on
glycolysis for the production of ATP
• maintenance of the biconcave, flexible
shape of the cell,
allowing it to squeeze through narrow
capillaries
- a reduced rate of glycolysis leads to
decreased ATP
production
- >phagocytosis =>results in hemolytic
anemia
- Reduction of pyruvate to lactate
• formed by lactate dehydrogenase, is the
final product of
anaerobic glycolysis in eukaryotic cells
• formation of lactate is the major fate for
pyruvate in lens and
cornea of the eye, kidney, medulla, testes,
leukocytes and RBC
- because these are poorly vascularised
and/or lack
mitochondria
• lactate formation in muscle:
- in exercising muscle NADH production exceeds the oxidative
capacity of the
respiratory chain
- elevated
NADH/NAD^+ ratio,
favouring
the reduction of
pyruvate to lactate
- lactate
accumulates in
muscle, the
intracellular pH
drops
• potentially resulting in cramps
• lactate eventually diffuses into
bloodstream
Page 52 91 of
• lactate consumption:
- the direction of the lactate reaction depends on the relative
 concentrations of pyruvate and lactate, and the ratio of NADH/
NAD^+ in the cell
• lactic acidosis:
- elevated concentrations of lactate in plasma
- occurs if there is a collapse of the circulatory system
• myocardial infarction
• pulmonary embolism
• uncontrolled hemorrhage
• shock
- failure to bring adequate amounts of oxygen into the tissues •
resulting in an impaired oxidative phosphorylation and
decreased ATP synthesis
• to survive the cell uses anaerobic glycolysis as a backup
system for generating ATP, producing lactic acid
-Energy yield from glycolysis
•anaerobic glycolysis: two molecules of ATP are
generated for each molecule of glucose converted to
two molecules of lactate
-there is no production nor consumption of NADH
•aerobic glycolysis: gain of two ATP per molecule of
glucose, two molecules of NADH are also produced

-administration of insulin initiates an increase in the

amount of glucokinase, phosphofructokinase, and
pyruvate kinase in liver
•>increase in gene transcription => increased enzyme
synthesis, favouring the conversion of glucose to
pyruvate
•>synthesis of glucokinase, phosphofructokinase, and
pyruvate kinase are decreased, when plasma glucagon
is high and insulin is low
(fasting diabetes)

-Alternative fates
of pyruvate
•oxidative
decarboxylation of
pyruvate
—>into acetyl CoA
fuel for TCA cycle
•carboxylation of
pyruvate to
oxaloacetate
•Reduction of
pyruvate to
ethanol
(microorganisms)
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Page 54 91 of
Page 55 91 of

Seminar No.10: Metabolism Of Carbohydrates II Oxidative

Decarboxylation Of Pyruvate.
Tricarboxylic Acid Cycle. Pentose Phosphate Pathway.

I. Oxidative decarboxylation of pyruvate:

A. Pyruvate dehydrogenase complex
B. Mechanism
C. Regulation
II. Tricarboxylic Acid (TCA) Cycle
A. Biochemical significance
B. Mechanism and energetic balance
C. Regulatory enzymes
D. Net energetic yield of glucose degradation in aerobic conditions III.
Pentose Phosphate Pathway (PPP)
A. Biological importance and stages
B. Glucose-6-phosphate dehydrogenase deficiency

- TCA It is the final pathway where the oxidative metabolism of

carbohydrates, amino acids, and fatty acids converge, their carbon
skeletons being converted to CO2
• Energy for the production of the majority of ATP in most animals
- TCA happening in mitochondria
- aerobic pathway, because O2 is required as the final electron acceptor •
functions in the formation of glucose
• provides building blocks for the synthesis of some amino acids and heme -
Reactions of the TCA cycle
• Oxidative decarboxylation of pyruvate
- Pyruvate must be transported into the mitochondrion (by specific pyruvate
transporter)
- in the matrix pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase
complex (PDH Complex)
• three enzymes
- pyruvate dehydrogenase (PDH or E1 or decarboxylase)
- dihydrolipoyl transacetylase (E2)
- dihydrolipoyl dehydrogenase (E3)
• regulatory enzymes are tightly bound
- pyruvate dehydrogenase kinase
- pyruvate dehydrogenase
• five coenzymes carrying or oxidant for the intermediates of the reactions -
E1 requires thiamine pyrophosphate (TPP)
- E2 requires lipoic acid and CoA
- E3 requires FAD and NAD^+
• regulation of the complex by covalent modification by the 2 regulatory enzymes -
activate and inactivate E1 (PDH)
- cyclic AMP-independent PDH kinase phosphorylates —>inhibits E1 -
PDH phosphatase dephosphorylates and activates E1
- kinase itself is allosterically activated by ATP, acetyl CoA, and NADH - >in the
presence of these high-energy signals, the PDH kinase, if pyruvate
concentrations are elevated, E1 will be maximally active
Page 56 91 of
 • calcium is a strong activator of the PDH phosphatase,
stimulating E1 activity
• >E.g.: skeletal muscle: CA^+ during contraction stimulates
the PDH complex and thereby energy production
• Pyruvate dehydrogenase deficiency:
- rare deficiency in the E1 component of the PDH complex
 - causing lactic acidoses
- inability to convert pyruvate to acetyl CoA
- >pyruvate is shunted to lactic acid via lactate dehydrogenase
- brain relies on TCA cycle for most of its energy & very
sensitive to acidosis
• neurodegeneration, muscle spasticity and in neonatal
onset form early death
• x-linked ,affects males and females (dominant)
• no treatment
• Mechanism of arsenic poisoning
- interfere with glycolysis at the glyceraldehyde 3-phosphate
step, decreasing the ATP production
- inhibition of enzyme that require lipoic acid as a coenzyme
including E2 of the PDH complex, alpha-ketoglutarate
dehydrogenase and branched chain chain alpha-leto acid
dehydrogenase
- arsenide forms a stable complex with the thiol(-SH) groups of
lipoic acid —>unavailable to serve as a coenzyme
• pyruvate accumulates, causing neurologic disturbance and
death
• synthesis of citrate from acetyl CoA and oxaloacetate
- condensation of acetyl CoA and oxaloacetate to form citrate is
catalysed by citrate sythethase
- this aldol condensation has an equilibrium far in the direction of
citrate synthesis
- citrate synthase is not an allosteric enzyme,
• inhibited by its product, citrate

Page 57 91 of
• substrate availability is another means of regulation
for citrate synthase
- binding of oxaloacetate causes a conformational
change in the enzyme that generates a binding site
for acetyl CoA
• Isomerisation of citrate
- citrate is isomerism to isocitrate by aconitase
• aconitase is inhibited by fluoroacetate (used as rat
poison) • fluoroacetate is converted to fluoroacetyl
CoA, which
condenses with oxaloacetate to form fluorocitrate
(potent inhibitor of aconitase —> citrate
accumulates
• oxidation and decarboxylation of isocitrate
- isocitrate dehydrogenase catalyses the irreversible
oxidative decarboxylation of isocitrate yielding the first
of three NADH molecules and releases CO2
- allosterically activated by ADP and Ca^+, and inhibited
by ATP and NADH
 • Oxidative decarboxylation of alpha-ketoglutarate
- the conversion of alpha-kltogluterate to succinyl CoA is
catalysed by the alpha-ketoglutarate dehydrogenase complex -
producing the second NADH of the cycle
- releasing CO2
- coenzyme required are thiamine pyrophosphate, lipoic acid,
FAD, NAD^+, and CoA
- alpha-ketoglutarate complex is inhibited by its products, NADH
and succinyl CoA, and activated by Ca^2+
- >BUT not regulated by phosphorylation/dephosphorylation
reactions as described for PDH complex
• cleavage of succinyl CoA
- succinate thiokinase cleaves the high-energy thioester bond of
succinyl CoA
- this reaction is coupled to phosphorylation of guanosine
diphosphate (GDP) to guanosine triphosphate
- GTP and ATP are energetically interconvertible by the
nucleoside diphosphate kinase reaction
- > GTP+ADP<——>GDP+ATP
• Oxidation of succinate
- oxidised to fumarate by succinate dehydrogenase,
as FAD is reduced to FADH2
- enzyme functions as complex II of the electron chain
• Hydration of fumarate
- fumarate is hydrated to malate in a freely reversible
reaction catalysed by fumaras
- Fumarate is also produced in the Urea cycle
• oxidation of malate
- malate is oxidised to oxaloacetate by malate
dehydrogenase - produces third and final NADH
- deltG° of the reaction is positive, but the reaction is
driven in the same direction of oxaloacetate by highly exergonic citrate
synthase - Energy produced by the TCA cycle
• 2 carbon atoms enter the cycle as acetyl CoA and leave as CO2 •
the cycle does not involve net consumption or production
oxaloacetate or of any other intermediate

Page 58 91 of
• four pairs of electrons are transferred during the
cycle - three pair reducing three NAD^+ to NADH
- one pair reducing FAD to FADH2
• oxidation of one NADH by the electron transport
chain leads to formation of approximately three
ATP
• oxidation of one FADH2 yields approximately two
ATP - Regulation of the TCA cycle
• controlled by the regulation of several enzyme
 activities - most important of these regulated enzymes are
those that catalyse reactions with highly negative deltaG° •
citrate synthase
• isocitrate dehydrogenase
• alpha-ketoglutarate dehydrogenase complex -
reducing equivalents needed for oxidative
phosphorylation are generated by the pyruvate
dehydrogenase complex and the TCA cycle, and both
processes are upregulated in response to a rise in ADP
Page 59 91 of
Page 60 91 of
Pentose Phosphate Pathway
- also called the hexose monophosphate pathway, or 6-
phosphogluconate pathway
- in the cytosol of the cell
- 2 irreversible oxidative and a series of reversible sugar
phosphate reactions
- no ATP is directly consumed or produced in the cycle
- direction and rate are determined by the supply and demand of
intermediates of cycle
- Irreversible oxidative reaction
• three reactions that lead to the formation of ribulose 5-
phosphate, CO2, and two molecules NADPH for each
glucose 6-phosphate oxidised
• dehydrogenation of glucose 6-phosphate
- catalysed by glucose 6-phosphate dehydrogenase
- oxidation of glucose 6-phosphate to 6-
phosphogluconolactone
- NADP^+ is the coenzyme
- regulated primarily at the G6PD reaction NADPH is a
potent competitive inhibitor
• increased demand for NADPH, the ratio of NADPH/
NADP^+ decreased and flux through the cycle increases
in response to the enhanced activity of G6PD
• insulin up regulates expression of the gene for G6PD
- increases in well fed state
• Formation of ribulose 5-phosphate
Page 61 91 of
- 6-Phosphogluconolactone is hydrolysed by 6-phosphogluconolactone hydrolase -
irreversible and not rate-limiting
- oxidative decarboxylation of the product, 6-phosphogluconate dehydrogenase -
irreversible reaction produces a pentose sugar-phosphate (ribulose 6-phosphate),
CO2, and a second molecule of NADPH
- Reversible non oxidative reactions
• reactions catalyse the interconversion of sugars
containing
3-7 carbons
• these reversible reactions permit ribulose
5-phosphate to be
converted to ribose 5-phosphate or to
intermediates of
glycolysis - fructose 6-phosphate and
glyceraldehyde 3-
phosphate
• if cells have a greater need for NADPH than for
ribose 5-
phosphate, transketolase and transaldolase
convert the
ribulose 5-phosphate produced as an endproduct of the
oxidative reactions to glyceraldehyde 3-phosphate and
fructose 6-phosphate, which are intermediates of glycolysis
 • non oxidative reactions can provide the biosynthesis of
ribose 5-phosphate from glyceraldehyde 3-phosphate and
fructose 6-phosphate in the absence of the oxidative steps
- Uses of NADPH
• phosphate group on one of the ribose units
• in the cytosol of hepatocytes the steady-state ratio of NADP^+/NADPH is approximately
0.1 which favours the use of NADPH in reductive biosynthetic reactions - favours an
oxidative role for NAD^+
• Reductive biosynthesis
- NADPH a high-energy molecule, like NADH,
- electrons of NADPH are destined for the use in reductive biosynthesis, rather than the
transfer to oxygen as is the case with NADH
- part of the energy of glucose 6-phosphate is conserved in NADPH, a molecule with a
negative reduction potential can be used in reactions requiring an electron donor

• reduction of hydrogen peroxide

- hydrogen peroxide is one of a family of reactive oxygen species (ROS) formed from the
partial reduction of molecular oxygen
• formed continuously as by-products of aerobic metabolism, through reactions
with drugs and environmental toxins, or when the level of antioxidants is
diminished, all creating the condition of oxidative stress
- >can cause serious chemical damage to DNA, Proteins, and unsaturated lipids,
and can lead to cell death
Page 62 91 of
- enzymes that catalyse antioxidant reactions:
• reduced glutathione, can chemically detoxify hydrogen
peroxide
- catalysed by the selenium-requiring glutathione
peroxidase, forms oxidised glutathione, which no longer
has protective properties
- the cell regenerates reduced glutathione in a reaction
catalysed by glutathione reductase, using NADPH as a
source of reducing equivalents
- additional enzymes such as superoxide dismutase and
catalase, catalyse the conversion of other toxic oxygen
intermediates to harmless products
- Antioxidant chemicals
• ascorbate
• vitamine E
 • beta-carotene
• —>able to reduce and, thus, detoxify oxygen intermediates
in the laboratory
• but failed to show clear beneficial effects
- Cytochrome P450 monooxygenase system
• in the cytochrome P450 monooxygenase
system, NADPH
provides the reducing equivalents required by
this series of
reaction
->

R-H+O2+NADPH+H^+——>R-OH+H2O+NADP^+
- >>R may be a steroid, drug, or other chemical
• Mitochondrial system
- biosynthesis of steroid hormones
• hydroxylate intermediates in the conversion of
cholesterol to steroid hormones, making these
hydrophobic compounds more water soluble liver uses
this same system in bile acid synthesis and in the
hydroxylation of cholesterol to 25-hydroxycholecalciferol,
and the kidney uses it to hydroxylate vitamin D3 to its to
its biologically active 1,25-dihydroxylated form
• Microsomal system
- membranes of the smooth endoplasmic reticulum is the
detoxification of foreign compounds (xenobiotics)
- numerous drugs and pollutants as petroleum products and
pesticides
- >hydroxylate these toxins using NADPH as the source of
reducing equivalents
• first activate or inactivate a drug or second make a toxic
compound more soluble —>facilitating its excretion in
 the urine or feces
- the new hydroxyl group will serve as a site for
conjunction with a polar molecule, such as gluronic
acids, which will significantly increase the compound’s
solubility
• Phagocytosis by wight blood cells
- phagocytosis is the ingestion by receptor-mediated endocytosis of microorganisms,
foreign particles, and cellular debris by cells such as neutrophils and macrophages
(monocytes)
Page 63 91 of
- Oxygen-independent mechanism
• use pH changes in phagolysosomes and lysosomal enzymes
to destroy pathogen
- Oxygen-dependent system
• include the enzymes, NADPH oxidase and myeloperoxidase
(MPO)
• the bacterium is recognised by the immune system and
attacked by antibodies
• NADPH oxidase is activated and reduces molecular oxygen
from the surrounding tissue into superoxide (O2^-), a free
radical ->”respiratory burst”
• flavocytochrome plus additional peptides
• electrons move from NADPH to O2, via FAD and heme,
generating (O2^-)
• genetic deficiencies in NADPH oxidase cause chronic
granulomatous disease severe, persistent infections and
formation of granulomas
• superoxide is converted to hydrogen peroxide (a ROS) or
catalysed by superoxide dismutase (SOD)
• peroxide plus chloride ions are converted to hypochlorous acid
(bleach) —>kills bacteria
- peroxide can also be partially reduced to the hydroxyl
radical (OH), or be fully reduced to water by catalase or
glutathione peroxidase
• Synthesis of nitric oxide (NO)
- mediator in a broad array of biologic systems
- relaxing factor, which causes vasodilation by relaxing vascular
smooth muscle
- can act as a neurotransmitter, prevents platelet aggregation, and
plays an central role in macrophage function
- often confused with nitrous oxide (N2O) “laughing gas”
- has a very short half life in tissues, because it reacts with
oxygen and superoxide, and then is converted into nitrates and
nitrites including peroxynitrite (O=NOO^-), a reactive nitrogen
species (RNS)
- Synthesis of NO
• arginine, O2, and NADPH are substrates for
cytosolic NO
synthase
- Flavin mononucleotide (FMN), flavin adenine
 dinucleotide
(FAD), heme, and tetrahydrobioptrin are coenzymes
- NO and citrulline are products of the reaction
- three NO synthases have been identified
• two are constitutive (synthesised at a constant rate
regardless of physiological demand), calmodulin
dependent enzymes
- found primarily in endothelium (eNOS), and neural
tissue (nNOS)
• inducible, Ca^2+ - independent enzyme (iNOS) are expressed in many cells,
e.g. hepatocytes, macrophages, monocytes, and neutrophils
• specific inducers for iNOS vary with cell type, and include tutor necrosis
factor-alpha, bacterial endotoxins and inflammatory cytokines
- >these compounds have been shown to promote synthesis of iNOS
Page 64 91 of
- actions of NO on vascular endothelium
• mediator in the control of vascular smooth muscle tone
• NO is synthesised by eNOS in endothelial cells
- activating the cytosolic form of guanylate cyclase (membrane associated) to
form cGMP
• rise in cGMP causes activation of protein kinase G, phosphorylating Ca^2+
channels causing decreased entry of Ca^2+ into smooth muscle cells
• >decreasing the calcium-calmodulin activation, of myosin-light chain kinase
• >>decreasing smooth muscle contraction and favouring relaxation of
vascular smooth muscle==> lowering blood pressure
• Note: sildenafil citrate, used in the treatment of erectile dysfunction, inhibits
the phosphodiesterase that inactivates cGMP
- Role of NO in mediating macrophage bactericidal activity
• iNOS activity is normally low
• synthesis is stimulated by bacterial lipopolysaccharide and gamma-interferon
release in response to infection
• activated macrophages form superoxide radicals that combine with NO to form
intermediates to decompose, forming the highly bacterial OH radical
- other functions of NO
• potent inhibitor of platelet aggregation by activating the cGMP pathway
• also characterised as a neurotransmitter in the brain
- Glucose 6-P Dehydrogenase deficiency
• inherited disease characterised by hemolytic anaemia caused by an inability to
detoxify oxidising agents
• X-linked
• a clinical manifestation of G6PD deficiency is neonatal jaundice appearing 1-4 days
after birth
- jaundice typically results from increased production of unconjugated bilirubin
• stickle cell trait and beta-thalassemia minor also confer resistance
• Role of G6PD in red blood cells:
- NADPH that is essential for the maintenance of the reduced glutathione pool
- decreased in the cellular detoxification of free radicals and peroxides
- Glutathione also helps maintain the reduced states of sulfhydryl groups in proteins
 including hemoglobin
- >oxidation of those sulhydryl groups leads to the formation of denatured proteins,
forming insoluble masses “Heinz Bodies”
- causing RBC to be rigid, they are removed from circulation by macrophages

Page 65 91 of
• Precipitation factors in G6PD
deficiency
- Oxidant drugs:
• commonly used drugs
producing
haemolytic anaemia in
patients with
G6PD deficiency are best
remembered
from the mnemonic
AAA—Anitibiotics
(e.g.: sulfamethoxazole and
chloramphenicol) Antimalarials and
Antipyretics (e.g.: acetanilid)
- Favism:
• some forms of G6PD deficiency, for
example the mediterranean variant
(class II deficiency) are particularly
susceptible to the haemolytic effect of the lava (broad)
bean
- Infection:
• inflammatory response to infection results in the
generation of
the free radicals in macrophages, which can diffuse into RBC and
cause oxidative damage
• Properties of the variant enzymes
- many enzymes show altered kinetic properties
• decreased catalytic activity and stability
• alteration of binding affinity for NADP^+, NADPH, or glucose 6-P
- correlates with the amount of residual enzyme activity in the
 patients RBC
• Molecular biology of G6PD
- 400 mutations in the gene
- > most that are resulting in enzymic deficiency are missense mutations - both G6PD A^-
and G6PD mediterranean represent mutant enzymes that differ from the respective
normal variants by a single amino acid
- complete absence of G6PD activity is lethal
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Seminar No.11: Metabolism of carbohydrates III

Gluconeogenesis, Glycogenogenesis and Glycogenolysis.

1. Gluconeogenesis - mechanism and substrate,

thermodynamic barriers
2. Reciprocal regulation of gluconeogenesis and
glycolysis
3. Glycogenogenesis - mechanism.
Glycogenolysis -
mechanism
4. Regulation of glycogen metabolism:
1. allosteric effect on regulatory enzymes
2. covalent modification, the impact of
hormones
5. Defects in glycogen metabolism. Glycogenoses.
6. Regulation of blood glucose levels.
7. “Experimental work:” Estimation of blood
glucose curve
of health subject and diabetic patient.

1.
- During a prolonged fast glucose is formed from
precursors such as lactate, pyruvate,
alpha-ketoacids
- 90% of gluconeogenesis occurs in the liver, 10%
kidneys
• during prolonged fasting kidneys contribute
about 40%
of total glucose production
- Substrates for Gluconeogenesis include
intermediates of
glycolysis and the tricarboxylic acid (TCA) cycle
• Glycerol, lactate, alpha-keto acids,
- Glycerol is released during the hydrolysis of
triacylglycerols in adipose tissue, it is
phosphorylated by glycerol kinase to
glycerol
phosphate which is
oxidised by
glycerol phosphate
dehydrogenase to
dihydroxyacetone phosphate
 -Lactate is released into blood
by exercising skeletal muscle,
by cell that lack mitochondria
(RBC), in cori cycle:
•blood borne glucose is
converted by exercising muscle
to lactate, which diffuses into
blood
•lactate is taken up by the liver
and reconverted to glucose which is released back in
circulation
-Amino acids such as alpha-keto-glutarate are derived from the
metabolism of glycogenic amino acids, can enter the TCA cycle
and form oxaloacetate (OAA), a direct precursor to
phosphoenolpyruvate
Page 68 91 of
- Reactions unique to gluconeogenesis: 7 glycolytic reactions are reversible (used in
synthesis of glucose from lactate or pyruvate) BUT 3 are irreversible (must be
circumvented by 4 alternate reactions
• Carboxylation of of pyruvate
- “first roadblock” —> pyruvate is first carboxylated by pyruvate carboxylase to OAA,
which is then converted to PEP by the action of PEP-carboxykinase
• Biotin is a coenzyme, Pyruvate carboxylase requires covalently bound to the ϵ
amino group of a lysine residue in the enzyme-biotin-CO2 intermediate
• >>carboxylates pyruvate to form OAA

• Pyruvate carboxylase is allosterically activated by acetyl CoA

- elevated levels of acetyl CoA in mitochondria signal a metabolic state in which
the increased synthesis of OAA is required
 - at low levels of acetyl CoA pyruvate carboxylase
• Transport of oxaloacetate to the cytosol
- it must first be reduced to malate by mitochondrial
malate dehydrogenase, can be transported from
mitochondria to cytosol where it is deoxidised to
oxalocetate by cytosolic malate dehydrogenase as
NAD+ in reduced, the NADH produced is used in the
reduction of 1,3-BPG to glycolysis and
gluconengenesis
• decarboxylation of cytosolic oxaloacetate
- oxaloacetate is decarboxylated and phosphorylated to
PEP in cytosol by PEP-carboxykinase (reaction is
driven by hydrolysis of guanosine triphosphate)
• dephosphorylation of fructose 1,6-bisphosphate
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- hydrolysis of fructose 1,6-bisphosphate by
fructose 1,6-
bisphosphatase bypasses the irreversible
phosphofructokinase-1 reaction and
provides an
energetically favourable pathway for the
formation of
fructose 6-phosphate
- regulation by energy levels within the cell
• Fructose 1,6-bisphosphatase in inhibit by
elevated
adenosine levels , high levels of ATP and
low
concentrations of AMP stimulate
gluconeogenesis
- regulation by fructose 2,6-bisphosphatase
- Fructose 1,6-bisposphatase is inhibited by fructose 2,6-
bisphosphate, an allosteric effector whose concentration
is influenced by the level of circulating glucagon
• Dephosphorylation of glucose 6-phosphate
- hydrolysis of glucose 6-phosphate by glucose 6-
phosphatase bypasses the irreversible hexokinase
• requires for release of free glucose from glucose 6-
phosphate glucose 6-phosphate translocase, which transports glucose 6-
phosphate across the endoplasmic reticulum (ER), the ER enzyme glucose 6-
phosphatse removes the phosphate
• >deficiency of glucose
6-phosphatase “severe
fasting hypoglycaemia
because free glucose
is unable to be produced from
either
gluconeogenesis or
glycogenolysis
- Regulation of gluconeogenesis
• primarily determined by circulation
 level of
glucagon and availability of gluconeogenic
substances
• —>Glucagon
this hormone from the alpha cells of pancreatic
islets stimulates gluconeogenesis by three
mechanisms
1. changes in allosteric effectors: Glucagon lowers
the level of fructose 2,6-bisphospate, resulting
in activation of fructose 1,6-bisphosphatase and
inhibition of phosphofructokinase-1, thus
favouring gluconeogenesis over glycolysis
2. covalent modifications enzyme activity:
Glucagon G protein-coupled receptor and, via
an elevation in cyclic AMP (cAMP) level and
cAMP-dependent protein kinase activity,
stimulates the conversion of hepatic pyruvate
kinase to its inactive form, this decreases the
conversion of PEP to pyruvate, which has the
effect of diverting PEP to the synthesis of
glucose
3. Induction of enzyme synthesis: Glucagon
increases the transcription of the gene for EPE
carboxykinase, thereby increasing the
availability of this enzyme as levels of this rise
during fasting
• insulin causes decreased transcription of the
Page 70 91 of
mRNA for this enzyme
• Substrate availability
- decreased levels of insulin favour mobilisation of amino acids for muscle protein -
provide carbon skeletons for gluconeogenesis
- ATP and NADH, coenzymes-cosubstrates required for gluconeogenesis, are
primarily provides by catabolism of fatty acids
• Allosteric activation by acetyl CoA
- allosteric activation of hepatic pyruvate carboxylase
by acetyl
CoA occurs during fasting
- increased lipolysis in adipose tissue
- liver is flooded with fatty acids
- rate of formation of acetyl CoA by beta-oxidationof
these fatty
acids exceeds the capacity of the liver to oxidise it to
CO2 and
H2O
- acetyl accumulates and pyruvate carboxylase is
activated
• Allosteric inhibition by AMP
- fructose 1,6-bisphosphatase is inhibited by AMP — a
compound that activates phosphofructokinase-1
- >reciprocal regulation of glycolysis and
gluconeogenesis
Glycogen Metabolism
- a constant source of blood glucose is an absolute requirement for
human life
- blood glucose can be obtained from diet by degeneration of
glycogen and gluconeogenesis
• intake of glucose and glucose precursors (starch,
monosaccharides, and disaccharides)
- storing a supply of glucose in a rapidly mobilised form
(glucogen) • can be rapidly released from liver and kidney
glycogen (to
maintain blood glucose concentration
• fuel reserve for the synthesis of adenosine
triphosphate (ATP)
during muscle contraction
- 400g glycogen make up 1-2% of fresh weight of a resting
muscle - 100g glycogen make up 10% of fresh weight of a
well-fed adult liver - Structure and function of glycogen:
• Glycogen branched-chain polysaccharide made
exclusively from alpha-D-glucose
- primary glycosidic bond is an alpha(1->4) linkage
- after 8-10 glucosyl residues there is a branch
containing an
alpha(1->6) linkage
- >>exit in discrete cytoplasmic granules
• Liver glycogen stores increase during the well-fed state
- muscle glycogen is not affected by short periods
- synthesised to replenish muscle stores after they have
been
depleted following strenuous exercise
- Synthesis of glycogen (Glycogenesis)
• Glycogen is synthesised from alpha-D-glucose
• requires energy supplied by ATP and uridine triphosphate
(UTP) • synthesis: occurring in cholesterol
- alpha-d-glucose attaches to uridine diphosphate, the
source of
all the glucosyl residues that are added the the growing
glycogen molecule
Page 71 91 of
- UDP-glucose is synthesised from glucose 1-phosphate
and UTP
by UDP-glucose pyrophosphorylase
- the high energy bond in Phosphate (PPi), second
product of the
reaction is hydrolysed into two inorganic phosphates by
pyrophosphatase
- glycogen synthase is responsible for for making the
alpha(1->4)
linkages
 • can only elongate already existing chains
• a fragment of glucose can serve as a primer
• glycogenic can serve as an acceptor of glucose residues from UDP-glucose • side
chain hydroxyl group of specific tyrosine serves as a site at which the initial glucose
from UDP-glucose
- producing a short alpha(1->4)-linked glucosyl chain
• this short chain serves as a primer
• can be elongated by glycogen synthase
• >elongation of glycogen chain involves transfer of glucose from UDP-glucose to
the non reducing end of the growing chain
- forming a new glycosidic bond between the anomeric hydroxyl of carbon 1 of
the activated glucose and carbon 4 of the accepting glucose residue
- enzyme responsible for making the alpha(1->4) linkages in glycogen is
glycogen synthase
• UDP + ATP <——> UTP + ADP

- glycogen has branches located eight glucosyl residues apart (highly branched,
tree-like structure
• more stable than unbranched amylose
• branching also increases the number of nonreducing ends to which new glucosyl
residues can be added and the site of the molecule
• accelerating the rate at which glycogen synthesis can occur

Page 72 91 of
• Branches are made by the action of the
branching
enzyme, amylo-alpha(1->4) to alpha (1->6)-
transglycosidase
- removing a chain of 6-8 glucosyl residues
from the
 nonreducing end
- breaking an alpha(1->4) bond to another residue
on the chain and attaches it to a non-terminal
glucosyl residue by an alpha(1->6) linkage;
functioning as 4:6 transferase
- >can be further elongated now by glycogen
synthase
• additional branches:
- terminal 6-8 glucosyl residues can
be removed
and used to make additional
branches
- the degenerative pathway that mobilises
stored glycogen is
not a reversible reaction
• glycogen phosphorylase sequentially
cleaves the alpha(1-
>4) glycosidic bonds
• by simple phosphorolysis (producing
glucose 1-
phosphate) until four glucosyl units
remain on each chain
before a branch point
• removal of branches:
- oligo-alpha(1->4)->alpha(1->4)-glucan
transferase
activity removes the outer three of the
four glucosyl
residues attached at a branch
- transferring them to the nonreducing
end of another
chain, lengthening it accordingly
• an alpha(1->4) bond is broken and an
alpha(1-4)bond is made and the enzyme functions as
4:4 transferase
- the remaining single glucose residue attached in an
alpha-(1->6) linkage is removed hydrolytically by
amylo-alpha(1->6)-glucosidase activity
• releasing free glucose
- the glycosyl chain is available again for degradation by
glycogen phosphorylase
• Glucose 1-phosphate, produced by glycogen phosphorylase is converted in the
cytosol to glucose 6-phosphate by phosphoglucomutase
- producing glucose 1,6-biphosphate as a temporary but essential intermediate • in
the liver glucose 6-phosphate is transported into the endoplasmic reticulum by
glucose 6-phosphate translocate
• than converted to glucose by glucose 6-phosphatase
• glucose moves to the ER of the cytosol
• hepatocytes release glycogen derived glucose into the blood to help maintain blood
glucose levels, until the gluconeogenic pathway actively produces glucose • in the muscle
glucose 6-phosphate can not be dephosphorylated because of a lack of glucose
6-phosphatase
• 1-3% of glycogen is continuously degraded by the lysosomal enzyme,
alpha(1-4)glucosidase (acid maltase)
- a deficiency of this enzyme, causes accumulation of glycogen in vacuoles in the
lysosomes,
• resulting in the serious glycogen storage disease Type II: Pompe disease
Page 73 91 of
Page 74 91 of
- Regulation of glycogen synthesis and degradation
• in the liver glycogenesis accelerates during periods when the body has been fed well,
whereas glycogenolysis accelerates during periods of fasting
• in skeletal muscle glycogenolysis occurs during active exercise
- glycogenesis begins as soon as the muscle is at rest again
• glycogen synthase and glycogen phosphorylase are hormonally regulated
- meeting the needs of the body as a whole
• pathways of glycogen synthesis and degradation are allosterically controlled
- to meet the needs of specific tissues

Page 75 91 of
• Activation of glycogen degradation by
cAMP-directed
pathway
- glucagon or epinephrine, that bind to the G
protein
coupled receptors (GPCR) signals the need for
glycogen
to be degraded
- Activation of protein kinase A
• G protein-mediated activation of adenylyl
cyclase
 • this enzyme catalyses the synthesis of cAMP,
activating cAMP-dependent protein kinase A
• PKA is a tetramere, with two regulatory subunits (R)
and two catalytic subunits (C)
- cAMP binds to the regulatory subunit dimer,
releasing individual catalytic subunits that are active
- PKA than phosphorylates several enzymes of
glycogen metabolism
- activation of phosphorylase kinase
• phosphorylase kinase can be inactive (b form) or
active (a form)
- PKA phosphorylates the inactive form which is
becoming active
- inactivated by hydrolytic removal of its phosphor
group by protein phosphatase-1
• this enzyme is activated by a signal cascade
initiated by insulin
• insuline also activates the phosphodiesterase that
degrades cAMP
- Activation of glycogen phosphorylase (two forms)
• dephosphorylated inactive b form
• phosphorylated active a form
• active phosphorylase kinase phosphorylates glycogen
phosphorylase b to its active a form, beginning
glycogenolysis
• phosphorylase a is reconverted to phosphorylase b by
the hydrolysis of its phosphate by protein
phosphatase-1
• Inhibition of glycogen synthesis by a cAMP-directed
pathway
- regulated enzyme in glycogenesis is glycogen synthase (two forms) •
the active form a is dephosphorylated
• the inactive form is phosphorylated
• Allosteric regulation of glycogen synthesis or degradation
- glycogen synthase and glycogen phosphorylase respond to the levels of
metabolites and energy needs of the cell
- glycogenesis is stimulated when substrate availability and energy levels are high •
glycogenolysis is increased when glucose and energy levels are low - allows a rapid
response to cell needs
- can override effects of hormone-mediated covalent regulation
- regulation of glycogen synthesis and degradation in the well fed state • glycogen
synthase b in liver and muscle is allosterically inhibited by glucose 6- phosphate as
well as by ATP
• in liver, not in muscle, non-phosphorylated glucose is also an allosteric inhibitor of
glycogen phosphorylase a

Page 76 91 of
- activation of glycogen
degradation
 by calcium
• Ca^2+ is released into the
cytoplasm
- in muscle in responds to neural
stimulation
- in liver in responds to
epinephrine binding to alpha1-
adreneric receptors
- Ca^2+ binds to calmodulin,
triggering a conformational
change such that the activated
- calmodulin functions as an
essential subunit of many
complex proteins
• one such protein is
phosphorylase kinase b,
activated by the
Ca^2+calmodium complex
• Epinephrine at beta
adrenergic receptors signal
through a rise in cAMP, not
calcium
- ATP for muscle contractions is
supplied by the degeneration of muscle glycogen to
glucose , which can than enter glycolysis
• nerve impulses cause membrane deploarisation
- promoting Ca^2+ release
- from sarcoplasmic reticulum
- Ca^2+ binds calmodulin and the complex activates
muscle phosphorylase kinase b
- epinephrine released from the adrenal medulla signals
need for blood glucose
• epinephrine to hepatocyte alpha-adrenergic G protein
coupled receptors activates a phospholipid-dependent
cascade
- resulting in movement of Ca^2+ from the ER into the
cytoplasm
- Ca^2+calmodulin complex forms and activates hepatic
phosphorylase kinase b
- muscle phosphorylase is active in the presence of the high AMP concentration
that occur in the muscle under extreme conditions of anoxia and ATP depletion •
AMP binds to glycogen phosphorylase b causing its activation without
phosphorylation
•
Page 77 91 of
Seminar No.12: Metabolism Carbohydrates IV Metabolism
Of Mannose, Fructose, Galactose And
Lactose

1. Metabolism of mannose
2. Metabolism of fructose-enzyme and disturbances in fructose
metabolism
3. Metabolism of galactose-enzymes and disturbances in
galactose metabolism
4. Synthesis of lactose in mammary glands

- Source of Fructose is the disaccharide sucrose

• when cleaved in the intestine releases euquimolar amounts
of fructose and glucose
• the entry of fructose in not insulin dependent
• to enter the pathway of intermediate metabolism it must be
phosphorylated by fructokinase or hexokinase(in most cells)
- low affinity for fructose (high Km)
- fructokinase provides the primary mechanism for fructose
phosphorylation
• found in liver, kidney, and small intestinal mucosa
• converting fructose to fructose 1-phosphate, using ATP
as a donor
• Cleavage of fructose 1-phosphate
- is not phosphorylated to fructose 1,6-bisphosphate as is
fructose 6-phosphate, but is cleaved by aldolase B to
Page 78 91 of
dihydroxyacetone phosphate (DHAP) and glyceraldehyde
• Aldolase A (in most tissues)
• Aldolase B (in liver) only one that cleaves fructose 1-
phosphate
• Aldolase C (in brain)
- >all cleave fructose 1,6-bisphosphate
• A deficiency in enzymes required for the entry of fructose into
intermediate metabolic pathways can result in either a benign
condition as a result of fructokinase deficiency “fructosuria”
- disturbance of liver and kidney metabolism as a result of
aldolase B deficiency
- 1:20.000 live births
- appear when baby is weaned from milk and begins to be
fed food containing sucrose or fructose
- fructose 1-phosphate accumulates
• resulting in a drop in the level of inorganic phosphate (Pi)
(ultimately drop in ATP and AMP rises)
• in absence of Pi AMP is degraded
- causing hyperuricemia
• decreased availability of hepatic ATP affects
gluconeogenesis (causing hypoglycemia with vomiting),
and protein synthesis (decreased blood clotting factors)
• kidney function may also be affected
• HFI can be diagnosed by fructose in the urine
- fructose, sucrose and sorbitol must be removed from
the diet to prevent liver failure and possible death
Page 79 91 of
- individuals with HFI display an aversion to
sweets
and consequently have an absence of
dental caries
- Conversion of mannose to fructose 6-phosphate
• Mannose the 2-epimer of glucose
• hexokinase phosphorylates mannose, producing
mannose
6-phosphate
• (reversibly) isomerised to fructose 6-phosphate by
phosphomannose isomerase
• most intracellular mannose is synthesised from
fructose
- Conversion of glucose via sorbitol
• Aldose reductase reduces glucose, producing
sorbitol
(found in lens, retina, Schwann cells of peripheral
nerves,
liver, kidney, placenta, red blood cells, and cells in
ovaries
and seminal vesicles
• in cells of the liver, ovaries, seminal vesicles, is a
 second
second enzyme, sorbitol dehydrogenase
- can oxidise the sorbitol to produce fructose
- >sperm cells using fructose as a major carbohydrate
energy source
• the pathway from sorbitol to fructose in the liver provides a
mechanism by which any available sorbitol is converted
into a substrate that can enter glycolysis or
gluconeogenesis
• effect of hyperglycaemia on sorbitol metabolism:
- large amounts of glucose may enter these cells during
times of hyperglycaemia (e.g. uncontrolled diabetes)
- elevated intracellular glucose concentrations and an
adequate supply of NADPH cause aldose reductase to
produce a significant increase in the amount of sorbitol
which can not pass efficiently through cell membranes,
remaining trapped in the cell
- when sorbitol dehydrogenase is absent or low, sorbitol
accumulates in these cells, causing strong osmotic effect
• >cell swelling as a result of water retention
- The major dietary source of galactose is lactose obtained
from milk and milk products
• like fructose the entry of galactose into cells is not insulin
dependent
• galactose must be phosphorylated before it can be
metabolised further
- galactokinase produces galactose 1-posphate
• galactose can not enter the glycolytic pathway unless it is
first converted to UDP-galactose
- UDP-glucose reacts with galactose 1-phosphate, producing UDP-galactose and
glucose 1-phosphate
• the enzyme for this reaction is galactose 1-phosphate uridyltransferase (GLAT) -
for UDP-galactose to enter the mainstream of glucose metabolism, it must first be
converted to its C-4 epimer, UDP-glucose, by UDP-hexose 4-epimerase • UDP-galactose
can serve as the donor of galactose units in a number of synthetic pathways, including
synthesis of lactose, glycoproteins, glycolipids and glycosaminoglycans
Page 80 91 of
 - >if galactose is not provided E.g.: lack of beta-galactosidase
“lactose intolerant” all tissue requirements can be met by the
action of UDP-hexose 4-epimerase on UDP-glucose
• if GALT is deficient galactose 1-phosphate and galactose
accumulate in cells
- >the accumulated galactose is shunted into side pathways
such as that of galactitol production
• this reaction is catalysed by aldose reductase
- Lactose synthesis
• lactose is beta-galactose attached by a beta(1->4)
linkage to
glucose “milk-sugar”
• synthesised in Golgi by lactose synthase
(UDP-galactose:
glucose galactosyltransferase), which transfers
galactose from
UDP-galactose to glucose releasing UDP
- this enzyme is composed of two proteins
• Protein A is a beta-D-galactosyltransferase in a
number of
tissues other than the lactating mammary gland
- this enzyme transfers galactose from
UDP-galactose to
N-acetyl-D-glucosamine, forming the same
beta(1->4)
linkage found in lactose and producing N
acetyllactosamine
• Protein B found in lactating mammary glands
- it is alpha-lactalbumin, its synthesis is
stimulated by the
peptide hormone prolactin
- forms a complex with the enemy, protein A, changing the
specificity of that transferase so that lactose, rather than
N-acetyllactosamine, is produced