CHM4116: Amino Acid Biosynthesis Protein Turnover and Amino Acid Catabolism

INTRODUCTION

I. NITROGEN-CONTAINING COMPOUNDS
Amino Acids
• Building block of proteins
• Contains C, H, O, and N (in the form of NH4+)
• Can be obtained from degradation of proteins
o Cellular or dietary proteins
• Translation: metabolic pathway for protein synthesis
o Part of flow of genetic information
Porphyrins
• Synthesis of new protein requires source of amino acids
• Organic compounds found in globin molecules
• Amino acids are generated by digestion of dietary proteins
• Ex. myoglobin, hemoglobin
in the intestine and degradation in the cell
• Many cellular proteins are constantly degraded and
Nucleotides
synthesized = turned over
• Building blocks of nucleic acids
• Turning over of damaged or old proteins: recycling process
• Made up of sugar, phosphate, and nitrogeneous bases
• Cyclin-B = regulatory protein in cell mitosis/division
• 2 types of nitrogeneous base: purines and pyrimidines
o In metaphase, it is very visible but degraded
during anaphase to help develop complex
II. NITROGEN METABOLISM
system for turning over of proteins
- Protein Turnover - Purine Biosynthesis
• Damaged proteins whose accumulation can affect
- Amino Acid Deamination - Purine Catabolism
organism, is degraded
- Urea Cycle - Pyrimidine Biosynthesis & Catabolism
o Before it is degraded, it is first marked
- Breakdown of Amino Acids - Conversion of ribonucleotides to
- Amino Acid Biosynthesis dexoyribonucleotides o ATP-dependent process: ubiquitin mark proteins
- Nitrogen Fixation - Conversion of dUTP to dTTP for degradation
- Heme Synthesis o Poly-ubiquitinated proteins are eventually
digested by proteasome
• Poly-ubiquitinated proteins must have at least 4 ubiquitin
Protein Turnover
molecules for it to be digested by 26-S proteasome
• Balance, degradation, and synthesis of proteins
• Cellular proteins are constantly turning over: rate depends
on their functions AMINO ACID CATABOLISM
o Regulatory function = short lifespan
Amino Acid Deamination
• When amino acids are released (degradation of protein),
they can serve as precursor for protein synthesis and
source of N for synthesis of other compounds
• Excess not stored: catabolized as source of metabolic fuel
• 1st step: deamination – removal of ammonium ion (NH4+)
Urea Cycle
• NH4+ processed and converted to urea: secreted as waste
material • Amino acids provided through degradation or digestion
Breakdown of Amino Acids are used as building blocks for synthesis of proteins and
• When amino acid is deaminated, only C skeleton remains other nitrogenous compounds (ex. nucleotide bases)
• Depending on amino acid, it can release different C • Excess amino acids no longer needed are catabolized to be
skeleton = different pathways used as metabolic fuel
Nitrogen Fixation
• Source of N for biosynthesis = atmospheric N (cannot be 1st Step: Deamination
readily assimilated into organic systems) • Amino group is released and processed in urea cycle
Conversion of dUTP to dTTP • Amino acid → urea → excreted through urine
• Carbon skeleton → major metabolic intermediates that
• Uracil differ from thymine in terms of structure
can be used as a source of energy → completely converted
• Presence of methyl group at C5
to CO2 and water or metabolite for CAC
• First to be synthesized = UTP
• Converted to TTP by simple methylation reaction • Other proteins, once marked with several ubiquitin
molecules, are degraded by proteasome
Amino Acids Obtained Through Diet
• Essential: better that is acquired through diet, since their
synthesis is long
• Ex. arginine: requires 20 rxns for it to be synthesized
• Ex. alanine - synthesized by transamination rxn of pyruvate
• Although mammals synthesize arginine, they cleave most
of it to form urea
o An enzyme hydrolyzes arginine to ornithine and
urea (urea cycle)
Digestion of Dietary Protein
• Acidic character of stomach favors protein denaturation
o Denatured proteins = easier to digest
• Pepsin: primary enzyme in stomach
o Non-specific protease, optimum pH is 2
• Digestion continues in lumen of intestine
• Pancreatic enzymes: trypsin, chymotrypsin, elastase
• Mixture of different proteases with different specificity
allow digestion of dietary proteins to amino acids and
short chain of peptides
• From lumen, they are transported to intestinal cell, where
they are further digested to amino acids and transported
via bloodstream, to be released to different tissues
Degradation of Cellular Proteins
• Proteins have specific lifespan
• Half-life of enzyme varies depending on the tissue
• Long-lived enzymes are more stable
o Their activity is constant under different
physiological conditions
• Short-lived enzymes are involved in metabolic control
o Quantity depends on physiological need of
organism
Cells Synthesize Proteins from and Degrade them to Amino Acids
• To store nutrients in the form of proteins and to break in
times of metabolic need
• To eliminate abnormal proteins whose accumulation
would be harmful to the cell
• To permit the regulation of cellular metabolism by
eliminating superfluous enzymes and regulatory proteins
o Eliminate enzymes that exceed amount needed
PROTEIN DEGRADATION
I. LYSOSOMES: Degrade Many Proteins
• Contain ~50 hydrolytic enzymes, including a variety of
proteases: cathepsins
• Maintain an internal pH of 5: serves as protection against
accidental breakage of lysosome, which can leak lysosomal
enzymes
o Optimum pH: acidic side
o Not active in cytosolic side (pH at physiological
condition)
• Degrade substances that the cell takes up via endocytosis
• Recycle intracellular constituents that are enclosed within
vehicles that fuse with lysosomes in a process called
autophagy
• In a well-nourished cell, lysosomal protein degradation is
non-selective
o Some essential enzymes and regulatory proteins
can be lost – alarming when cell is starving
• In starving cell, a selective pathway is activated after a
prolonged fast that import and degrades cytosolic proteins
containing KFERQ sequence
o Protein containing KFERQ sequence would be
lost in tissues that are sensitive to fasting (ex.
liver, kidney)
II. UBIQUITIN
• For protein breakdown for eukaryotic system,
independent of lysosomal degradation
• Ubiquitin = ubiquitous = always present
o In eukaryotic protein, it is the one of the most
conserved proteins
• Marks proteins for its degradation
• 76-residue protein present in all eukaryotic ells
• A tag that marks the protein for destruction
Process of Marking
• The carboxyl terminal glycine residue of ubiquitin becomes
covalently attached to the ɛ-amino groups of several lysine
residues on a protein to be degraded
• Ub-carbonyl = C terminal of ubiquitin
o Terminal amino acid = glycine
o Forms amide bond with ɛ-amino group of a
lysine residue of protein to be degraded =
isopeptide bond
• Protein to be degraded has many lysine residues: potential
sites for ubiquitination
Ubiquitination Reactions Cyclin Destruction Boxes
• Amino acid sequences that mark cell-cycle proteins for
destruction
Protein Rich in PEST sequences
• Proline, Glutamine, Serine, Threonine

PROTEASOME
In an ATP-requiring reaction, ubiquitin's terminal carboxyl groups is 26S Proteasome
conjugated, via thioester bond, to ubiquitin-activating enzymes (E1)

• A 26 S large protease complex digests ubiquitinated

The ubiquitin is then transferred to a specific Cys sulfhydryl group on proteins
one of the numerous homologous proteins named ubiquitin- • An ATP driven multi-subunit protease spares ubiquitin,
conjugating enzymes (E2s) which is then recycled
• Ubiquitin conjugated to 1st enzyme is transferred to • 2 components
another thiol group of another enzyme o 20S (catalytic unit) 19S (regulatory unit)

20S Proteasome

Ubiquitin-protein ligase (E3) transfer the activated ubiquitin from E2

to a lysine ɛ-amino of a previously bound protein = forms isopeptide • Has the catalytic unit
• E3 is bound initially to a protein to be degraded • Has 2 copies each of 14 homologous proteins
• Ubiquitin that is activated by E1 and E2 is transferred to • Arranged in 4 rings of 7 subunits that stack to form a barrel
another lysine residue of condemned protein (sealed)
• Outer rings: made up of α-subunits
Isopeptidases • Inner ring: β-subunits
• For protein to be degraded, it must be linked to a chain of o Contains active site
at least 4 tandemly-linked ubiquitin molecules o N-terminal of a specific β-subunit contains serine
• Ubiquitin molecules are removed (and recycled) by or threonine
ubiquitin isopeptidases o Serine and threonine contain OH: serves as
• After tagging, proteins are degraded by proteasome nucleophile which can attack carbonyl of the
o Proteasome would not degrade ubiquitin peptide
o Ubiquitin is reused for marking other proteins

SIGNALS TO IDENTIFY PROTEIN DESTRUCTION

N-Terminal Rule
• Determines whether protein gets ubiquitinated The proteasome and other
• The half-life of a cytoplasmic protein is determined to a proteases generate free amino
large extent by its amino terminal residue acids

Shows fates of amino acids that

are released by the proteins of
the lysosomal degradation

• Ex. if N terminal is Met, the lifespan of protein is > 20 hrs

AMINO ACID DEAMINATION
• Some amino acids are catabolized initially by deamination,
wherein amino group is converted to urea
• Most amino acids release their amino group and
transferred to α-ketoglutarate, to become glutamate
I. TRANSAMINASES: Use PLP to Transfer Amino Group
• Transamination: 1st step in amino acid catabolism, release
of amino group
• Aminotransferases catalyze the transfer of an α-amino
group from α-amino acid to an α-keto acid
• Transaminases generally funnel α-amino groups from a
variety of α-amino acids to α-ketoglutarate for conversion
into NH4+
• The predominant amino group acceptor is α-ketoglutarate,
producing glutamate and new α-ketoacid
• After α-ketoglutarate receives amino group from other
amino acids, it becomes source of amino group to other
keto acids
• Example: oxaloacetate is transaminated with amino group
from glutamate → aspartate
o Glutamate → α-ketoglutarate
• Glutamate's amino group can be transferred to
oxaloacetate, yielding aspartate and reforming α-
ketoglutarate
Coenzyme: Pyridoxal-5’-phosphate (PLP)
• These aminotransferases require pyridoxal-5'-phosphate
o A derivative pyridoxine (Vitamin B6)
o Forms a Schiff base
• Has a carbonyl group
• Attached to active site of enzyme by a Schiff base with a
Lys residue
• When amino group from source (ex. glutamate) is
transferred to PLP, it becomes pyridoxamine
• When enzyme has not facilitated transamination, PLP is
attached to active site of the enzyme via Schiff base,
formed between carbonyl group of PLP and ε-amino group
of a Lys residue
II. GLUTAMATE
• Transamination does not result to net deamination
o There are other pathways where glutamate can
be deaminated
• Can be oxidatively deaminated
• Glutamate Dehydrogenase (GDH): mitochondrial enzyme
o Can accept NAD+ or NADP+ as its redox coenzyme
II. SERINE AND THREONINE
Ser and Thr can be directly deaminated
Catalyzed by serine dehydratase and threonine
dehydratase
Serine dehydrated to form pyruvate
Threonine dehydrated to form 2-ketobutyrate
III. PERIPHERAL TISSUES Urea
• Transport nitrogen to the liver
• The nitrogen is first removed from the amino acid and
converted in a form that can be absorbed by the liver and
converted to urea
• Nitrogen is transported to the liver into 2 transport forms
• The muscle does not contain enzymes of urea cycle - must
have another way to deliver amino acid to the liver and for
liver to convert it to urea
• Synthesized in the liver by the enzymes of the urea cycle
[1] Glucose-Alanine Cycle
• Secreted into the bloodstream and sequestered by the
kidney for excretion in the urine
• N's of urea comes from the ammonium ion released by
amino acid and amino group of aspartic acid
o Carbonyl comes from bicarbonate
• Aspartic acid is released as fumarate after urea cycle = can
enter CAC
• 5 enzymatic reactions are involved in the urea cycle
o 2 mitochondrial, 3 cytosolic

Reactions

• In the muscle, after prolonged exercise, amino acids are

degraded to be used for cellular respiration
• Amino group is transferred to pyruvate
• Alanine is released into bloodstream, transported to the
liver, where it is converted back to pyruvate by
transamination
• In the liver, alanine is transaminated and amino group is
transferred to glutamate, where glutamate is oxidatively
deaminated to release NH4+ and converted to urea

[2] Nitrogen Transported as Glutamine

• Glutamine synthetase catalyzes the synthesis of glutamine
from glutamate and NH4+ in an ATP-dependent rxn
• NH4+ + glutamate + ATP → glutamine + ADP + Pi
• The N of glutamine can be converted to urea in the liver

IV. UREA CYCLE

• 5 reactions incorporate ammonia and an amino group into
urea
• The rate of the urea cycle changes with the rate of amino
acid breakdown

Ammonia and Less Toxic Waste Products

[1] Carbamoyl Phosphate Synthase Acquires 1st Urea N Atom

• Ammonium ion is converted to urea in most terrestrial

vertebrates
• Carbamoyl phosphate synthetase catalyzes condensation
of amino group and bicarbonate to form carbamoyl
phosphate
o Carbamoyl grp: carbonyl C attached to amino grp
• ATP-dependent rxn
[2] Ornithine Transcarbamoylase
• Transfer of carbamoyl to ornithine = citrulline
• Ornithine is formed in cytosol but rxn takes place in
mitochondria
• Ornithine must be transported via special transport
protein into the mitochondria
o Structure similar to Lys - Lys side chain is longer
• Citrulline, formed in mitochondria, is used in the cytosol =
must be transported also
o Looks like a precursor of arginine
• Ornithine and citrulline are non-standard amino acids
o Classified as amino acids but not found in
proteins
[3] Arginosuccinate Synthetase Acquires 2nd Urea N Atom
• Citrulline binds w/amino grp of aspartate= arginosuccinate
[4] Arginosuccinase Forms Fumarate and Arginine
• Arginosuccinate hydrolyzed by arginosuccinase to form
arginine and fumarate
• Fumarate can be converted to oxaloacetate or enter CAC
[5] Arginase Releases Urea
• Arginine hydrolyzed to release ornithine, which can be
transported to mitochondria and initiate another cycle of
urea cycle
Regulation of Urea Cycle
• Regulated by substrate availability
N-Acetylglutamate
• Synthesized from glutamate and acetyl-CoA by N-
acetylglutamate synthase
• Activates CPS I (carbamoyl phosphate synthetase I)
o Enzyme that catalyzes the regulatory rxn in urea
cycle
• If amino acid degradation increases, concentration of
glutamate increases
o N-acetylglutamate and CPS I activity increases
• As amino acid degradation increases, rate at which amino
group is secreted into system by urea cycle also increases
BREAKDOWN OF AMINO ACIDS
I. OVERVIEW
• Degradation of proteins can be through lysosomal
degradation or marking with ubiquitin and digestion by
proteasome
• Some amino acids are used as a metabolic fuel -
catabolized to be converted as a source of energy
• Standard amino acids are degraded to one of the seven
metabolite intermediates
o Pyruvate, α-ketoglutarate, succinyl-CoA,
fumarate, oxaloacetate, acetyl-CoA,
acetoacetate
• Once ammonium ion is converted to urea, it can already
be secreted
o Ammonium ion in the body is toxic
Division of Amino Acids Based on Catabolic Pathway
Glucogenic Amino Acids
• Degraded to pyruvate, α-ketoglutarate, succinyl-CoA
fumarate, or oxaloacetate and therefore, glucose
precursors
• Alanine, cysteine, glycine, serine, arginine, glutamate,
glutamine, histidine, proline, valine, methionine,
aspartate, asparagine
• Can be converted to glucose and excess can be stored as
glycogen
Ketogenic Amino Acids
• Broken down to acetyl-CoA or acetoacetate and thus
converted to fatty acids or ketone bodies
• Leucine, lysine: cannot be converted to carbohydrates
(purely ketogenic)
• Humans lack enzymes for converting acetyl-CoA to glucose
Both Glucogenic and Ketogenic
• Threonine, tryptophan, isoleucine, phenylalanine, tyrosine
II. Ala, Cys, Gly, Ser, Thr Degraded to Pyruvate
• Methylene group is released from another Gly and
oxidized when transferred to Gly
• When Gly is cleaved, methylene group is initially
transferred to carrier (THF - tetrahydrofolate)
o THF carries methylene between N5 and N10
Cysteine
• Thiol group is converted to pyruvate
• Thiol group released as hydrogen sulfide, sulfite, or
thiocyanate and ammonia
Threonine
• [1] conversion to glycine by serine
hydroxylmethyltransferase: removal of hydroxymethyl
group
• [2] major route: threonine catabolized by threonine
dehydrogenase, releases acetaldehyde and ketobutyrate
o Hydroxyl group of threonine oxidized to
carbonyl: α-amino-β-ketobutyrate
o Acted upon by lyase, releasing acetyl-group as
acetyl-CoA and glycine
III. Asn, Asp Degraded to Oxaloacetate

• Aspartate = removal of amino group = oxaloacetate

• Asparagine = amide group initially hydrolyzed by
asparaginase to become aspartate
o Aspartate becomes oxaloacetate by
transamination

IV. Arg, Glu, Gln, His, Pro Degraded to α-Ketoglutarate

Alanine
• Alanine → pyruvate (1 step) by transamination
• Transfer of amino group to α-ketoglutarate: glutamate
• α-keto acid of alanine is left = pyruvate
Serine
• Serine → pyruvate (1 step) by dehydration reaction
• Recall: aliphatic amino acids (with OH) directly deaminated
by dehydration rxn (serine and threonine)
• Initially forms aminoacrylate → pyruvate
• Threonine → 2-ketobutyrate
Glycine
• Must be first converted to serine by serine
hydroxylmethyltransferase
• Hydroxymethyl group is added
o Source: another glycine molecule acted upon by
glycine cleavage system
Glutamate
• Converted directly to α-ketoglutarate by transamination
• Can also be oxidatively deaminated by glutamate
dehydrogenase to become α-ketoglutarate
Glutamine
• Amide group hydrolyzed by glutaminase = glutamate
• Glutamate oxidatively deaminated
• Glutaminase helps in neutralizing acid by producing
ammonium ion, which serve as a buffer, during acidosis
Arginine
• Hydrolyzed by arginase, releases urea and ornithine
o Last step of urea cycle
• NH4+ of ornithine removed by transamination (ornithine-δ-
aminotransferase) = glutamate-5-semialdehyde
• Oxidation of glutamate-5-semialdehyde = glutamate → α-
ketoglutarate
Proline and Histidine
• Converted to semialdehyde or glutamate then α-
ketoglutarate by oxidative deamination
V. Met, Thr, Ile, Val Degraded to Succinyl-CoA
• During metabolism of odd-numbered FAs, the last acetyl-
CoA is propionyl-CoA
o Propionyl-CoA becomes succinyl-CoA
• Degradation of these amino acids yields propionyl-CoA,
then converted to succinyl-CoA
Methionine
[1] When Met is activated by its rxn with ATP, it becomes S-
adenosylmethionine (SAM)
• SAM: good methylating agent
• When rxn requires addition of methyl group, usual donor
is SAM
• Methyl group is originally from methionine
[2] biosynthetic methylation: methyl group of SAM is transferred to
acceptor = methylated acceptor
• When methyl group is donated to acceptor, SAM is
converted to S-adenosylhomocysteine
[3] When adenosine is released, it becomes homocysteine
[4] Homocysteine can be converted back to methionine by addition
of methyl group
Threonine
[5] homocysteine condenses with serine = cystathionine
[6] synthesis of cysteine from methionine and serine
• When cystathionine is broken down by lyase, it would lead
to α-ketobutyrate and cysteine
• α-keto acid dehydrogenase converts α-ketobutyrate to
propionyl-CoA then succinyl-CoA
VI. Branched-Chain Amino Acid Degradation
• Involves acyl-CoA oxidation
• Eventually, isoleucine and valine are converted to
propionyl-CoA by dehydrogenase rxn
o Uses FAD as oxidizing agent
VII. Leu, Lys Degraded to Acetyl-CoA and/or Acetoacetate IX. Phe, Tyr Degraded to Fumarate and Oxaloacetate
Lysine

NITROGEN FIXATION
I. INTRODUCTION
• Many amino acids are synthesized by pathways that are
present only in plants and microorganism

Flow of Nitrogen in the Biosphere

• An 11 rxn pathway which starts with formation of the α-

ketoglutarate-lysine adduct saccharopine
• Rxn 4: PLP-dependent transamination
• Rxn 5: oxidative decarboxylation of an α-keto acid by a
multienzyme complex similar to pyruvate dehydrogenase
• Rxn 6, 8, 9: standard rxns of fatty acyl-oxidation =
dehydrogenation of FAD, hydration, dehydrogenation by
NAD+
• Rxn 10, 11: standard rxns in ketone body formation
• Rxn 5, 7: 2 mol of CO2 are produced • The atmosphere is a rich source of inorganic nitrogen
• Atmospheric nitrogen (N2) not highly reactive but must be
VIII. Trp Degraded to Alanine and Oxaloacetate converted to ammonia or nitrates to be biologically useful
to plants and animals
• Enzymes involved in nitrogen fixation are present in
bacteria = nitrogen-fixing bacteria
• Ammonia can be converted to nitrate by nitrifying bacteria
• Some nitrogen return to atmosphere by denitrifying
bacteria, in exchange for oxygen

Nitrogen Fixation
• Tryptophan eventually converted to alanine and
acetoacetate
• Tryptophan can also form pyruvate
• The process by which inorganic molecular nitrogen (N 2) in
the atmosphere is incorporated first into ammonia, and
then into organic compounds that are of use to organisms
• Reduction of N2 to NH3
• Bacteria are responsible for reduction and form symbiotic
relationships that result in nodules on the roots of
leguminous plants
• Reduction is catalyzed by the nitrogenase enzyme complex
• Nitrogen from the atmosphere incorporated into
biologically useful compounds through nitrogen fixation
o Microorganisms use ATP and a powerful
reductant to reduce atmospheric nitrogen to
ammonia
II. REDUCTION OF N2 TO NH3
• Carried out by some bacteria and archaea (60%)
• Lightning and UV radiation (15%)
• Industrial processes (25%)
o Haber-Bosch system: for preparing fertilizer
o Problem: expensive since reduction of N and H
to NH3 would have to take place at a very high
temperature and pressure

Nitrogenase Complex

• Nitrogen fixation is carried out by a complex of enzymes

with high reducing powers = nitrogenase complex
• An enzyme present in Rhizobium bacteria that live in root
nodules of leguminous plants
• Catalyzes production of ammonia from molecular nitrogen
• Some free-living soil and aquatic bacteria possess
nitrogenase
• Has reductase (Fe protein) and nitrogenase (MoFe protein)
o Both contain iron-sulfur complex
• Reductase: provides high-reducing electrons, which comes
from reduced ferredoxin
o Transfers electrons to nitrogenase
o Uses hydrolysis of ATP to effectively transfer
electrons
• Nitrogenase: use electrons to reduce N2 to NH3
Reaction
N2 + 8H+ + 8 e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi
Per transfer of e-, it requires hydrolysis of 2 ATP
Summary
• Nitrogen enters the biosphere by the process of nitrogen
fixation
• Atmospheric nitrogen is converted to NH3 in its conjugate
acid form, NH4+
• Nitrogenase enzyme catalyzes crucial rxns in nitrogen
fixation
III. FEEDBACK INHIBITION: Nitrogen Metabolism
• If there is a high level of end product amino acid or
nucleotide, the cell saves energy by not making the
compound through a feedback mechanism
o End-product serves as competitive inhibitor of
the enzyme catalyzing 1st rxn
• In summary, since biosynthetic pathways for many
nitrogen-containing compounds are long and complex,
feedback inhibition helps save energy
• Glutamine syntethase: catalyzes synthesis of glutamine
from glutamate in presence of NH4+ and ATP
o Inhibitors: histidine, serine, carbamoyl
phosphate, AMP, tryptophan, CTP
• Nucleotides also serve as inhibitor of glutamine synthetase
(AMP, CTP)
o These amino acids are raw materials for
synthesis of nucleotides
o Surplus of nucleotides = inhibitor
IV. ASSIMILATION OF NITROGEN INTO BIOMOLECULES
• NH4+ is assimilated into amino acids through glutamate
and glutamine
• α-amino group of most amino acids comes from glutamate
• Glutamine contributes its side chain (N) for the
biosynthesis of wide range of important compounds (ex.
tryptophan and histidine)
• Common features of amino acid biosynthesis include:
transamination and 1C transfers
o 1C carrier: tetrahydrofolate (THF)
o Most common methyl carrier: S-
adenosylmethionine (SAM)
• Glutamate is formed by reductive amination of α-
ketoglutarate and NH4+
o Amination of glutamate = glutamine
o Used for synthesis of other amino acids due to
existence of an enzyme that can synthesize
glutamate from α-ketoglutarate and NH4+
• All amino acids are grouped into families based on their
biosynthetic pathways
Schiff Base Formation AMINO ACIDS: From Citric Acid Cycle and Other Major Pathways

• Amino donor reacts with carbonyl compound = Schiff base

• Reaction with water forms protonated Schiff base, which is
then hydrolyzed
• Cofactor needed to facilitate transamination: PLP
o Forms Schiff base with Lysine
• Carbonyl is provided by α-ketoglutarate
• Ammonium is fixed by nitrogen-fixing bacteria = forms
protonated Schiff base, which is hydrolyzed and reduced
to form glutamate
• Glutamate dehydrogenase: responsible for reductive
amination of α-ketoglutarate to form glutamate
• α-ketoglutarate and oxaloacetate = metabolites of CAC
• Pyruvate = gluconeogenesis and glycolysis
• Erythrose-4-P: pentose phosphate pathway
• Formation of Ribose-5-P = needed for histidine

I. Synthesis of Arg, Glu, Asn, Gln from Pyruvate, Oxaloacetate, and α-

ketoglutarate
• Glutamate, glutamine = amino acids that are derived from
α-ketoglutarate
• Alanine = from pyruvate
• In the presence of ATP, glutamate undergoes amidation to • Aspartate and asparagine = pyruvate
form glutamine
o Enzyme: glutamine synthetase
• 2nd ammonia forms amide with glutamate side chain =
glutamine
o Requires ATP

Prokaryotes: Synthesis of Glutamate

• Glutamate dehydrogenase: forms glutamate from α-

ketoglutarate in the presence of NADPH and ammonium
ion
• Glutamine syntethase: glutamine is synthesized from • To convert the aspartate and glutamine to their
glutamate using ATP and ammonium ion corresponding amides
o Glutamate dehydrogenase and glutamine o Synthesis of asparagine by asparagine synthetase
synthetase = present in all organisms o Source of amino group side chain: glutamine
• Some prokaryotes have revolutionary enzyme: glutamate o Glutamine → glutamate
synthase
• Synthesis of glutamine by glutamine synthetase
o Uses NADPH with ATP and low ammonium ion
o Glutamate initially activated by ATP
o Synthesizes glutamate from α-ketoglutarate with
o Source of amino group: 2nd ammonium ion
ATP and glutamine
o Amino group is provided by glutamine
Glutamate: Precursor of Pro, Arg, and Ornithine
• Glycine cleavage System: participates only during
formation of serine
o Provides methylene group from cleavage of
glycine to be carried by THF, transferred to
glycine to become serine

[1] Direct conversion of serine to glycine by serine

• Comparing with catabolism of arginine and proline (and
histidine), they are catabolized in α-ketoglutarate
• Arginine: in catabolism, arginine is acted upon by arginase
to release urea and ornithine, acted upon by ornithine-δ-
aminotransferase to release amino group to α-
ketoglutarate to glutamate = semialdehyde is formed
o In biosynthesis: glutamate is converted to
semialdehyde → transaminated to form
ornithine → arginine
• Proline: from glutamate, non-enzymatic rxn that leads to
cyclization of semialdehyde, producing it to form proline
3-Phosphoglycerate: Precursor of Ser, Cys, Gly
hydroxymethyltransferase in a rxn that yield N5,N10-methylene-THF
• Methylene group transferred from serine to THF and
released as N5,N10-methylene-THF, becoming glycine
[2] Condensation of N5,N10-methylene-THF with CO2 and NH4+
• Glycine synthase: uses CO2 and NH4+ to synthesize glycine
• Reaction: CO2 + NH4+ + N5,N10-methylene-THF + NADH ↔
Gly + THF + NAD
Cysteine (For Animals)
• Synthesized from serine and homocysteine
• Homocysteine longer by 1 methylene group than cysteine
• Cysteine and serine differ in replacement of S with O
o Cysteine and methionine: both contain S, but
Met has 2C attached to the α-carbon before the
thioether group

• Comparing its structure to an amino acid, it is an α-carbon

• Missing to be considered as amino acid: amino group
• Its hydroxyl is replaced by amino group
• For hydroxyl to be transaminated, it must be oxidized

3PG to Serine
• Serine is acetylated to form O-acetylserine
• The source of sulfur in plants and bacteria differ from that
of animals
• Sulfur donor comes from 3'-phospho-5'-adenylylsulfate
• Undergoes transamination to form 3-phosphoserine (PAPS)
o For a functional group to be phosphorylated, it
must be a hydroxyl
o Phosphate is hydrolyzed = serine
• **Serine directly dehydrated to pyruvate by serine
dehydratase

Serine to Glycine • Serine is acetylated to form O-acetylserine

• Involves transfer of 1C unit (N5'-N10'- • Acetate is replaced by sulfur = cysteine
methylenetetrahydrofolate)
• 1C acceptor: tetrahydrofolate (THF), from folic acid
• Serine participates in glycine synthesis in 2 ways
 • Participation of THF in conversion of serine to glycine
• Methylene group derived from serine is transferred to THF

PLANTS AND MICROORGANISMS: Synthesize Essential Amino Acids

• Adenylate is phosphorylated at 3' and 5'

o Sulfated phosphate attached to 5'
• PAPS is reduced in plants and bacteria
o Sulfite is released and another reduction
occurs = sulfide
o Sulfide serves as S atom for conversion of
serine to cysteine

Tetrahydrofolate (THF)

Lys, Met, Thr Synthesis from Aspartate

• Carries activated 1C units at several oxidation levels

• Has 3 components: pteridine ring, p-aminobenzoate,
glutamate
• Atoms involved in carrying 1C units: N5 of pteridine
ring and N10 of p-aminobenzoate
o N5 and N10 = sites where methylene unit is
attached

1C Groups Carried by THF

• Differ in oxidation states • Aspartate must be initially activated = becomes aspartyl-β-

phosphate
• Methyl = 1 unbound e-
o Once phosphorylated, using ATP as source of
• If THF carries 1C groups through its N5 and N10 = it would
phosphate = name of enzyme ends with -kinase
only occupy N5
• Formation of β-aspartate-semialdehyde = can now
o Methylene intermediate has 2 binding sites =
proceed with formation of lysine, methionine, and
linked to THF through N5 and N10
threonine
o Semialdehyde is first converted to homoserine
Methionine
• Essential amino acid, cannot be produced in animals
• Reacts with ATP to form S-adenosylmethionine (SAM)
• SAM: good carrier/major donor of methyl group
o Once methyl group and adenosyl is released, it
becomes homocysteine
Leu, Ile, Val Derived from Pyruvate
• Branched amino acids are derived from pyruvate
o Source of amino group: glutamate
• Pyruvate group is converted to hydroxyethyl-TPP
o Hydroxyethyl-TPP condenses with another
pyruvate to form α-acetolactate
o Forms leucine and valine
• For isoleucine, hydroxyethyl-TPP condenses with α-
ketobutyrate
Aromatic Amino Acids (Phe, Trp, Tyr) Synthesized from Glucose
Derivatives
• Starts with condensation of 2 metabolites: PEP and E4P =
2-Keto-3-deoxyarabino-heptulosonate-7-phosphate (7C)
o C1 is a carboxylate = uronic acid
• Central metabolite: chorismate
o Branches into 2 pathways, forming tyrosine or
phenylalanine
o For tryptophan, indole (from chorismate and
glutamine) condenses with serine
Synthesis of His: Includes Intermediates in Nucleotide Biosynthesis
• Starts from formation of activated ATP - N1-N5-
phosphoribosyl ATP (formed from PRPP and ATP)
• Histidine has N in its imidazole ring = provided by adenine
ring and amino group from glutamine
Summary
• 2 of the most important classes of rxns in the biosynthesis
of amino acids are transamination rxns and 1C transfers
• Glutamate and glutamine: principal donors of amino
groups in transamination rxns
o Glutamate: provides α-amino group
o Glutamine: side chain
• Carriers of 1C groups: biotin, SAM, derivatives of folic acid
• Non-essential amino acids are synthesized in 1-3 steps
• Essential amino acids are synthesized in 5-16 steps
• Excess amino acids are converted to fat and stored
• Diet that lacks high quality proteins results in breakage of
body protein
AMINO ACIDS AS PRECURSORS OF BIOMOLECULES Heme Biosynthetic Pathway

• Sphingosine = serine
• Histamine = histidine
• Thyroxine = tyrosine
• Epinephrine = tyrosine
• Serotonin = tryptophan
• Nicotinamide = tryptophan

I. GLUTATHIONE

• Formation of heme starts with 8 molecules of δ-

• A γ-glutamyl peptide that serves as a sulfuhydryl buffer aminolevulinate = pyrrole rings
and antioxidant
o γ-glutamyl = carboxylate in γ forms peptide bond Heme Degradation
with amino group of Cys
o Thiol can be oxidized = form disulfide with
another glutathione
o Can quench free radicals through its thiol group

II. PORPHYRINS

• Formation of heme degradation products biliverdin and

bilirubin is responsible for color of brusies
• Synthesized from glycine and succinyl-CoA
NUCLEOTIDE SYNTHESIS AND DEGRADATION
I. INTRODUCTION
• Nucleotide: building blocks of nucleic acids
o Serves as energy currency of cell
o When hydrolyzed, they provide energy in order
to push biological rxns forward
• First step in biosynthesis (in mammals): condensation of • There are several coenzymes in metabolic pathways that
glycine and succinyl-CoA to form δ-aminolevulinate has nucleotide components
o Catalyzed by δ-aminolevulinate synthase: a PLP o Ex. NAD
synthase enzyme present in mitochondria
SYNTHESIS OF PURINE BIONUCLEOTIDES
• Kinases convert IMP-derived AMP and GMP to ATP and Reaction 2
GTP
o IMP = inosine monophosphate, precursor for
AMP and GMP
o Monophosphates are converted to their
corresponding triphosphates by kinases
• Purine nucleotide synthesis is regulated by feedback
inhibition and feedforward activation
o Surplus of products = pathway is inhibited
• Balance between nucleotides since they are raw materials
for nucleic acid synthesis = to prevent addition of incorrect • Acquisition of purine N9
nucleotide into growing polynucleotide chain • Amino group is added, which replaces pyrophosphate by
• Salvage rxns convert purines to their nucleotide forms amidophosphoribosyl transferase
o Nucleotide turnover = cells will release bases (A, o Source of amino group: glutamine
C, T, G) and are salvaged
Reaction 3

• N1 of purine ring comes from aspartate amine

o C4, 5, 7 = from glycine
o Glycine is added to purine ring • Acquisition of purine atoms C4, C5, C7
• N3 and N9 = from glutamide amide • Addition of glycine
• C2, C8 = formate
• C6 = bicarbonate Reaction 4

• IMP = initially synthesized purine derivative

o Concludes that the starting material for
synthesizing nucleotides = ribonucleotide (not
• Acquisition of purine atom C8
free bases)
o Comparable to purines (A, G)
Reaction 5
• Synthesized through assembly of purine base on ribose-5-
phosphate (R5P) (not separately)
o Pathway is composed of 11 rxns
o R5P: platform of purine nucleotide

REACTIONS
Reaction 1

• Acquisition of purine atom N8

• Glutamine provides another source of N = 6C ring

• Activation of R5P by ribose phosphate pyrophosphokinase

Reaction 6 Reaction 11

• Formation of purine imidazole ring

Reaction 7
• Cyclization to form IMP

SUMMARY
• R10 - R11 and R7 & R8 = catalyzed by a multifunctional
enzyme
• R3, R4, R6 = take place on a single protein
• Intermediate products of these multifunctional enzymes
are not readily released to the medium but are channeled
to the succeeding enzymatic activities of the pathway
• Acquisition of C6
IMP: Converted to Adenine and Guanine Ribonucleotide
Reaction 8

• Acquisition of N1

Reaction 9

AMP
1. Aspartate's amino group linked to IMP = adenylosuccinate
a. Carbon skeleton of Asp released as fumarate
2. Adenylosuccinate lyase eliminates fumarate = AMP
GMP
1. IMP is dehydrogenated to form xanthosine
monophosphate (XMP)
• Elimination of fumarate a. IMP dehydrogenase: oxidizes IMP to XMP
b. Oxidizing agent: NAD
Reaction 10 2. Gln amide nitrogen is transferred = GMP

Mycophenolic Acid

• Immunosuppressant
• Inhibits IMP dehydrogenase following kidney transplant

• Acquisition of C2
NUCLEOSIDE DIPHOSPHATES AND TRIPHOPHATES • Source of N: NH4+
• Synthesized by phosphorylation of nucleotide • Carbamoyl synthetase II: cytosolic enzyme, uses glutamine
monophosphates as source of N
• NDPs synthesized from corresponding NMPs by base • Rxn uses 2 ATP
specific nucleoside monophosphate kinases o [1] Source of phosphate for formation of product
o [2] Energy for condensation rxn
AMP + ATP ↔ 2ADP Adenylate kinase
Reaction 2
GMP + ATP ↔ 2GDP Guanylate kinase

• NDPs are converted to corresponding NTPs by

corresponding nucleoside diphosphate kinases
o GTP + ATP ↔ GTP + ADP

Purines: Can Be Salvaged (Conversion to Nucleotide Form)

• Adenine phosphoribosyltransferase (APRT) mediates AMP
formation using PRPP
o Adenine + PRPP ↔ AMP + Ppi
• Hypoxanthine-guaninephosphoribosyltransferase
(HGPRT) catalyzes the analogous rxn for both
hypoxanthine and guanine
o Hypoxanthine + PRPP ↔ GMP + Ppi
o Guanine + PRPP ↔ GMP + PPi
SYNTHESIS OF PYRIMIDINE RIBONUCLEOTIDES
I. INTRODUCTION
• Synthesis of carbamoyl aspartate by aspartate
transcarbamoylase (ATCase)
• Aspartate donates its amino group
• *Feedback inhibition: last product of a pathway is the
inhibitor of the enzyme catalyzing initial rxns
• Aspartate transcarbamoylase: inhibited by UMP
Reaction 3

• Atomic origins: pyrimidines

• Pyrimidine is assembled first, then added to R5P
o **Purines: purine ring assembled on R5P
Pyrimidine Ring
• N1, C4, C5, C6 = from aspartate
• Almost the whole molecule of aspartate is incorporated in
pyrimidine ring
• N1 = α-amino group of aspartic acid
• C6 = α-carbon of aspartic acid (has carboxylate attached) • Carbamoyl aspartate undergoes ring closure by
dehydration rxn, which forms dihydroorotate by
II. SYNTHESIS OF UMP: 6 STEPS dihydroorotase
• UMP is the precursor of CMP
• The pyrimidine ring is coupled to R5P after the ring has Reaction 4
been synthesized

Reaction 1

• Synthesis of carbamoyl phosphate from glutamine,

bicarbonate, and water by carbamoyl phosphate
synthetase II • Oxidation of dihydroorotate by dihydroorotate
dehydrogenase to form orotate
• Carbamoyl phosphate synthetase: present in urea cycle
and found in mitochondria
• Urea cycle is a way of converting NH4+ released in
transanimation from catabolism of amino acids into urea
Reaction 5
• Acquisition of ribose phosphate moiety
• Formation of nucleotide by coupling orotate to PRPP to
form OMP (orotate phosphoribosyl transferase)
Reaction 6
• OMP decarboxylation to form UMP by OMP-decarboxylase
• UMP converted to its triphosphate by 2 consecutive
phosphorylation rxns by nucleotide kinase
III. FORMATION OF CTP
• Cytosine differs from uracil by substitution of keto group
with amine
• CTP formed by amination of UTP by CTP synthetase
• Animals: source of N is glutamine
o Bacteria: supplied directly by NH4+
• There is a certain rate at which cytosine spontaneously
deaminates
o When deaminated, it is converted back to uracil
o It will pair with A instead of G = possibility of
mutation
• Uracil forms AU base pair, cytosine forms GC base pair
IV. PYRIMIDINE NUCLEOTIDE BIOSYNTHESIS REGULATION
• In E.coli, the main regulatory enzyme is ATCase, which
forms carbamoyl aspartate
• Activated by ATP and inhibited by CTP
• For animals, main regulatory enzyme is carbamoyl
phosphate synthetase II
o Activated by ATP and inhibited by UTP
o Second-level regulation by orotate
phosphoribosyl transferase (activated by
carbamoyl phosphate, inhibited by UMP)
FORMATION OF DEOXYRIBONUCLEOTIDES
I. RIBONUCLEOTIDE REDUCTASE
• Converts ribonucleotides to deoxyribonucleotides (NDPs
to dNDPs)
• C2 in ribose reduced by ribonucleotide reductase
• Deoxyribonucleotides are synthesized from their
corresponding ribonucleotides by reduction of their C2'
position rather than de novo synthesis from deoxyribose-
containing precursors
• UDP converted to deoxyuridine-diphosphate
Regulation
• Ribonucleotide reductase is regulated by a complex
feedback network
• Deficiency of dNTP is lethal
o dNTP is a precursor for DNA synthesis
o If not present, it would lead to incomplete
synthesis of DNA
• Excess is mutagenic because the probability that a given
dNTP will be erroneously incorporated into a growing DNA
strand increases with its concentration relative to those of
other dNTPs
 • Mutagenic = can alter base sequence
o Point mutation: specific nucleotide is changed
o Frame shift mutation: can change reading frame
during translation
• dNTPs should be balanced since the chances of it being
incorporated instead of the correct one, is high

II. dNTP PRODUCTION

• The final step in the production of all dNTPs is the
phosphorylation of the corresponding dNDPs
o dNDP + ATP ↔ dNTP + ADP
• It is catalyzed by nucleoside diphosphate kinase, the same
enzyme that phosphorylates NDP
• After dNTP is produced, uracil is converted to thymine

III. dUMP METHYLATION → THYMINE Regeneration of THF

• The dTTP substrate for DNA synthesis is derived from
dUTP, which is hydrolyzed by dUMP by dUTP
diphosphorylase (dUTPase)
o dUTP + H2O → dUMP + PPi
• dUMP is methylated to generate dTMP
• TMP is phosphorylated to form dTTP
• DNA polymerase, enzyme responsible for synthesizing
DNA during replication, cannot distinguish between UTP
and dTTP
o If UTP is not hydrolyzed first, it can be
incorporated by the enzyme
o To prevent erroneous incorporation of UMP into • Thymidylate synthase catalyzes methylation of dUMP to
the DNA DTMP
o Methylene group reduced to methyl and
Difference Between RNA and DNA transferred to C5 of uracil → thymine
• Dihydrofolate reductase uses NADPH as reducing agent
o Necessary to revert DHF to THF
• THF can obtain its methylene group from serine
o Serine converted to glycine by serine
hydroxymethyl-transferase
o Regenerates N5-N10-methyelene-THF
• Incorporation of dTTP in growing nucleotide chain during
replication is crucial
o Thymidylate synthase: important enzyme for
synthesis of dTTP, which is necessary for DNA
Sugars synthesis
• RNA: ribose DNA: deoxyribose
Nitrogenous bases Inhibition of Thymidylate Synthase in Cancer Therapy
• RNA: A, U, C, G
• DNA: A, T, C, G
• T vs U: methyl group in C5, simple methylation rxn
Competitive inhibitor of
IV. THYMIDYLATE SYNTHASE thymidylate synthase
• Methylates dUMP to dTMP
• Source of methyl group: N5-N10-
methylenetetrahydrofolate
o Carbon is in the form of methylene must be
reduced to methyl group
o Becomes oxidized to dihydrofolate

Competitive inhibitor of
dihydrofolate reductase
NUCLEOTIDE DEGRADATION Purine Nucleotide Cycle
I. INTRODUCTION
• Purines are broken down to uric acid
• Uric acid may be further catabolized for excretion
• Pyrimidines are converted to CoA derivatives for
catabolism
• Nucleotides may be directly absorbed by intestinal mucosa
or further degraded to free bases and ribose or ribose-1-
phosphate by nucleosidases and nucleoside
phosphorylases
o Nucleoside + H2O → Base + ribose
o Nucleoside + Pi → Base + ribose-1-P
• This pathway functions in muscle to prime the citric acid
by generating fumarate (released from adenylosuccinate)
Nucleotide Metabolism Summary
• Problem in citric acid cycle: muscle has no enzyme for
anaplerotic rxns
o Generation of fumarate from purine nucleotide
cycle is important for proper function in muscle

Xanthine Oxidase: Mini-Electron Transport System

II. MAJOR PURINE CATABOLIC PATHWAYS

In Animals • Enol tautomer is the predominant form of uric acid

Degradation of Uric Acid

• All converge to xanthine and converted to uric acid by

xanthine oxidase
• IMP, XMP, GMP first acted upon by nucleotidase to form
their respective nucleoside
o Removal of R1P by purine nucleoside
phosphorylase (PNP)
• Adenosine cannot be acted upon by PNP - must be
deaminated first to inosine
Gout: Caused by Excess Uric Acid
• Gout: characterized by elevated levels of uric acid in body
fluid
• Manifestation is excruciating pain of sudden onset
• Caused by deposition of nearly insoluble sodium urate
• Sodium urate and/or uric acid may also precipitate in the
kidney and ureters
o Results to renal damage and urinary tract
obstruction

Xanthine Oxidase Inhibitor

• β-alanine → malonyl-CoA by aminotransferase

• Alleviates gout
• Allopurinol: analogue of hypoxanthine
• Xanthine oxidase can oxidize allopurinol = alloxanthine
o Cannot convert to uric acid, so the product stays
in the active site in the reduced form

III. MAJOR PYRIMIDINE CATABOLISM PATHWAY

• Cytidine deaminated to uridine by cytidine deaminase

before R1P is removed
o Releases uracil → dihydrouracil by dihydrouracil
dehydrogenase

• Dihydrouracil is converted to β-alanine