Derivatives of Amino Acids and
Metabolism of Nucleotides
CH353 January 29, 2008
Anabolic Role of the Citric Acid Cycle
Purines
X
Error:
glycine not
glutamate
provides
carbons for
purines
Biosynthesis of amino acids & derivatives from citric acid cycle intermediates
require anaplerotic reactions (red arrows) for replenishing metabolites
Derivatives of Amino Acids
• Porphyrins and Heme
– Glycine + Succinyl-CoA (animals)
– Glutamate (bacteria & plants)
• Non-ribosomal peptide synthesis
– peptidoglycan, antibiotics
– glutathione (glutamate + cysteine + glycine)
• Modified amino acids
– plant compounds, neurotransmitters, polyamines
• Nucleotide heterocyclic bases
– purines and pyrimidines
Biosynthesis of Heme
animals
heme
precursor
bacteria, plants
Biosynthesis of Heme
Genetic Deficiencies in Heme Biosynthesis
Catabolism of Heme
purple
Regulated step: 3 isozymes
green
yellow
Important serum antioxidant
Bile pigment
yellow (oxidized)
red-brown (reduced)
Reactions with Monooxygenases
• Use 2 reductants for O2 (mixed-function oxygenases)
– One reductant accepts an O atom
– Other reductant provides 2 H’s to the second O atom
• General Reaction:
AH + BH2 + O–O → A–OH + B + H2O
Biosynthesis of Nitric Oxide
• NO involved in intercellular signaling
• NO synthase (a mixed-function oxygenase)
– dimer, similar to NADPH cytochrome P450 reductase
– cofactors: FMN, FAD, tetrahydrobiopterin, Fe3+ heme
– catalyzes a 5 e- oxidation
Biosynthesis of Creatine
• metabolite for storage of high
energy transfer potential phosphate
– phosphorylated at high [ATP]
• amidinotransferase exchanges
amino acids
– glycine for ornithine
• 1 substrate and 1 product same as
for arginase reaction except
different amidino group acceptor
– glycine instead of water
• S-adenosylmethionine methyl donor
Biosynthesis of Glutathione
• reducing agent
(redox buffer)
• non-ribosomal
peptide synthesis
• carboxyl groups
activated with ATP
(acyl phosphate
intermediates)
Non-ribosomal Peptide Synthesis
• Microbial peptides are synthesized by multi-modular
synthases; similar to fatty acid biosynthesis
• Modular complexes of enzymes for recognition,
activation, modification and condensation of a specific
amino acid to the growing polymer
• Features use of unusual amino acids, D-enantiomers,
and non-α peptide bonds
• Peptidoglycans, antibiotics and ionophores
Reactions with Pyridoxal Phosphate
• Amino acid racemase reactions
L-alanine ↔ D-alanine
Inhibitors of alanine racemase:
Antibiotics – peptidoglycan biosynthesis
Biosynthesis of Plant Compounds
• phenylalanine, tyrosine,
tryptophan precursors for
plant compounds:
–
–
–
–
–
lignin (phenolic polymer)
indole-3-acetate (auxin)
tannins
alkaloids, e.g. morphine
flavors, e.g. cinnamon,
nutmeg, cloves, vanilla,
cayenne pepper
Reactions with Pyridoxal Phosphate
• Decarboxylase reactions
Histidine → Histamine + CO2
Ornithine → Putrescine + CO2
Biosynthesis of Neurotransmitters
Pathways involve decarboxylases
and mixed-function oxygenases
(monooxygenases)
Biosynthesis of Spermidine and Spermine
Pathway involves decarboxylases
and S-adenosylmethione alkylation
African Sleeping Sickness
• Caused by Trypanosoma brucei rhodesiense
• Vaccines are ineffective: repeated change of coat antigen
• Therapy based on inhibitor of polyamine biosynthesis
Mechanism of Ornithine Decarboxylase
Inhibition of Ornithine Decarboxylase
Ornithine
DMF-Ornithine
Study Problem
• Antihistamines are compounds that block histamine
synthesis or binding to its receptor
• Histamine is synthesized from histidine by a pyridoxal
phosphate dependent decarboxylase
• Design an antihistamine drug candidate, based upon the
mechanism for decarboxylation
• Show the structure and its proposed mechanism of action
Overview of Nucleotide Metabolism
• Nucleotide functions
–
–
–
–
Activated precursors for synthesis of RNA, DNA and cofactors
Activation of biosynthetic precursors
Energy for cellular processes
Signal transduction
• Biosynthetic pathways
– de novo synthesis of purines and pyrimidines
• differ in order of attachment of ribose to base
– salvage pathways
• reacting a base with activated 5-phosphoribose (PRPP)
Precursors for Nucleotide Biosynthesis
• 5-phosphoribosyl-1-pyrophosphate
ribose phosphate pyrophosphokinase
Ribose 5-phosphate + ATP → 5-phosphoribosyl-1-pyrophosphate + AMP
Precursors for Nucleotide Biosynthesis
Tetrahydrofolate (H4 folate)
derivatives
• N5,N10-methylene-H4 folate
– thymidylate biosynthesis
• N5-formyl-H4 folate
– purine biosynthesis
Precursors for Nucleotide Biosynthesis
• Amino Acids
– Glycine for purine biosynthesis
– Aspartate for pyrimidine biosynthesis
• Amino Acid Nitrogen
– α-amino group of aspartate (purines)
aspartate + [acceptor] + ATP → succinyl-amino-[acceptor] + ADP + Pi
succinyl-amino-[acceptor] → amino-[acceptor] + fumarate
– amide group of glutamine (purines, pyrimidines)
glutamine + [acceptor] + ATP → amino-[acceptor] + glutamate + ADP + Pi
Activation of Amino Acceptors
• carboxylate or carbonyl acceptor are activated with ATP
• acyl-phosphate or phospho-enol intermediates formed
• nucleophilic substitution of phosphate with amino group
O
R–C–O–
O
C–C–R
H
O
ATP
ADP
R–C–OPO3–2
OPO3–2
C–C–R
O
R’NH2
PO4–2
R–C–NHR’
NHR’
C–C–R
Biosynthesis of the Purine Ring
• Multi-step synthesis from many precursors
– (numbers indicate order of addition to purine ring from PRPP)
5
2
6
3
7
4
1
Purine Biosynthesis
1. glutamine-PRPP amidotransferase
•
•
•
glutamine donates amide nitrogen to
activated 5-phosphoribose (PRPP)
committed step for purine synthesis
product unstable t½ = 30 seconds
2. GAR synthetase
•
•
glycine carboxyl activated with ATP
Pi displaced; amide bond formed
3. GAR transformylase
•
N10-formyl tetrahydrofolate donates
formyl group to glycine amino group
4. FGAR amidotransferase
•
•
ATP activates carbonyl group
amidotransfer displaces Pi
Purine Biosynthesis
5. FGAM cyclase (AIR synthetase)
•
•
ATP activates carbonyl
cyclization of imidazole ring
in bacteria & fungi:
6. N5-CAIR synthetase
•
•
ATP activates HCO3carbamoylation of exocyclic amine
7. N5-CAIR mutase
•
transfer of carboxylate to ring
in higher eukaryotes:
6. AIR carboxylase
•
•
formation of only C-C bond
no cofactors or ATP required
Purine Biosynthesis
8. SAICAR synthetase
•
•
•
aspartate is amino donor
ATP activates carboxylate
aspartate amino replaces Pi
9. SAICAR lyase
•
•
•
fumarate is eliminated
steps 8 & 9 analogous to urea cycle
AICAR from histidine biosynthesis
10. AICAR transformylase
•
N10-formyl H4 folate donates formyl
group to glutamine-derived amine
11. IMP synthase
•
•
cyclization of second purine ring
ATP activation not required
Organization of Purine Biosynthetic Enzymes
• Purine biosynthesis organized
into multienzyme complexes
• In eukaryotes, multifunctional
proteins for:
– Steps 1, 3 & 5
– Steps 6a & 8
– Steps 10 & 11
• In bacteria, separate enzymes
associate in large complexes
• Channeling of intermediates
avoids dilution of reactants
Synthesis of Adenylate and Guanylate
• AMP synthesis uses GTP for activation; amine from aspartate
• GMP synthesis uses ATP for activation; amide from glutamine
Reciprocal Regulation:
• GTP for needed for
AMP synthesis
• ATP needed for
GMP synthesis
Regulation of Purine Biosynthesis in E. coli
Feedback Inhibition (negative)
• Inhibition of 1st step in common
pathway by IMP, AMP & GMP
• Inhibition of 1st step in branch
– AMP inhibits AMP synthesis
– GMP inhibits GMP synthesis
• Inhibition of PRPP synthesis by
phosphorylated end products
ADP, GDP and others
Reciprocal Regulation (positive)
• Requirements of:
– ATP for GMP synthesis
– GTP for AMP synthesis
Nucleotide Biosynthesis
Purine Biosynthesis
Pyrimidine Biosynthesis
• Hypoxanthine (a purine) is
assembled on the ribose 5phosphate → Inosinate (IMP)
• Precursors:
– PRPP
– Glycine
– H4 folate-formate (2)
– HCO3–
– Glutamine (amide-N) (2)
– Aspartate (amino-N)
• IMP → AMP
• IMP → XMP → GMP
• Orotate (a pyrimidine) is made
first then added to ribose 5phosphate → Orotidylate
• Precursors:
– Carbamoyl phosphate
• HCO3–
• Glutamine (amide-N)
– Aspartate
– PRPP
• Orotidylate → UMP → UDP →
UTP → CTP
Pyrimidine Biosynthesis
Carbamoyl Phosphate Synthetase II
•
•
•
cytosolic CPS II enzyme involved in pyrimidine biosynthesis
mitochondrial CPS I involved in arginine & urea synthesis
bacteria have single enzyme for both functions
Steps:
1. bicarbonate phosphate synthesis (1st activation)
2. carbamate synthesis (NH3 from glutamine hydrolysis)
3. carbamoyl phosphate synthesis (2nd activation)
Carbamoyl Phosphate Synthetase
Bacterial enzyme has 2 subunits
(blue & grey) with 3 active sites
joined by a substrate channel
(yellow wire mesh)
• 1st site: Glutamine releases NH4+
(glutamine in green)
• 2nd site: HCO3– is phosphorylated
with ATP and reacts with NH4+ to
form carbamate (ADP in blue)
• 3rd site: Carbamoyl phosphate is
synthesized by phosphorylating
carbamate with ATP (ADP in red)
Pyrimidine Biosynthesis
2. aspartate transcarbamoylase
•
•
•
activated carbamoyl group transferred
to amine group of aspartate
Pi displaced; amide bond formed
committed step in pyrimidine synthesis
3. dihydroorotase
•
cyclization of pyrimidine ring
4. dihydroorotate dehydrogenase
•
oxidation of C-C bond using NAD+
5. orotate phosphoribosyl transferase
•
•
•
pyrimidine ring (orotate) is transferred
to activated 5-phosphoribose (PRPP)
PPi lost; aminoglycan bond formed
analogous to pyrimidine salvage
Pyrimidine Biosynthesis
6. orotidylate decarboxylase
•
•
catalyzes synthesis of UMP
very efficient enzyme
7. uridylate kinase
•
nucleoside monophosphate kinase
specific for UMP
8. nucleoside diphosphate kinase
•
generic enzyme for (d)NDP’s
9. cytidylate synthetase
•
•
•
an amidotransferase
UTP is aminated using glutamine
carbonyl group is activated with ATP
to form acyl phosphate intermediate
Cytidine 5’-triphosphate (CTP)
Pyrimidine Biosynthesis Enzyme Complexes
• Eukaryotes have a multifunctional protein with the
first 3 enzymes in pyrimidine biosynthetic pathway
C
A
D
carbamoyl phosphate synthetase II
aspartate transcarbamoylase
dihydroorotase
• CAD has 3 identical polypeptides (Mr 230,000) each
with sites for all 3 reactions
Regulation of Pyrimidine Biosynthesis
• Feedback inhibition of 1st step
aspartate transcarbamoylase
(ATCase) by CTP
• Bacterial ATCase has:
– 6 catalytic subunits
– 6 regulatory subunits
• Allosteric inhibition:
– 2 conformations of ATCase:
active ↔ inactive
– binding of CTP to regulatory
subunits shifts conformation
active → inactive
– ATP reverses effect of CTP
Activation of Nucleotides
• Nucleoside monophosphate kinases
– specific enzyme for each base (e.g. adenylate kinase)
– nonspecific for ribose (ribose or 2’-deoxyribose)
ATP + NMP
ADP + NDP
• Nucleoside diphosphate kinase
– generic enzyme, nonspecific for base or ribose
– nonspecific for phosphate donor or acceptor
NTP + NDP
NDP + NTP
donor
acceptor
acceptor
donor
Nucleotides for DNA Synthesis
2 Modifications:
• ribonucleotides reduced to 2’-deoxyribonucleotides
NDP → dNDP
• uracil (uridylate) methylated to thymine (thymidylate)
dUMP → dTMP
Reduction of Nucleotides
• NDP is reduced to dNDP by
reduced form of ribonucleotide
reductase
• Oxidized form of ribonucleotide
reductase is reduced by either
glutaredoxin or thioredoxin
• Oxidized form of glutaredoxin
is reduced by glutathione
• Oxidized form of thioredoxin is
reduced by FADH2
• Oxidized glutathione and FAD
are reduced by NADPH
Regulation of Ribonucleotide Reductase
Ribonucleotide Reductase
(E. coli)
• Active sites are between each
R1 and R2 subunit
• Two R2 subunits each contain a
tyrosyl radical and a binuclear
Fe3+ cofactor
• Two R1 subunits each have
sites for enzyme activity and
substrate specificity
• The (d)NTP bound to substrate
specificity sites determines
which NDP is reduced to dNDP
Regulation of Ribonucleotide Reductase
Binding at activity regulatory sites: Binding at substrate specificity sites:
•
ATP activates enzyme
•
dATP inhibits enzyme
•
•
•
ATP or dATP: ↑dCDP ↑dUDP
dTTP: ↑dGDP ↓dCDP ↓dUDP
dGTP: ↑dADP ↓dGDP ↓dCDP ↓dUDP
Biosynthesis of Thymidylate
• Precursors for thymidylate (dTMP) synthesis may arise
from (d)CTP or (d)UTP pools
CTP
cytidylate
synthetase
nucleoside
diphosphate
kinase
UTP
uridylate
kinase
UMP
Cyclic pathway for conversion
of dUMP to dTTP
• Thymidylate synthase uses
N5,N10-Methylene-H4 folate as
both one-carbon source and
reducing agent
• Dihydrofolate reductase reduces
H2 folate → H4 folate with NADPH
• Serine hydroxymethyl transferase
reaction restores
N5,N10-Methylene-H4 folate
• Net reaction:
dUMP + NADPH + serine →
dTMP + NADP+ + glycine
Chemotherapeutic Agents
Inhibitors of glutamine
amidotransferases:
• Block purine & pyrimidine
biosynthesis
Inhibitors of thymidylate
synthesis:
• thymidylate synthase
• dihydrofolate reductase
Chemotherapy Targets
Group Study Problem
• Conversion of dUTP to dTTP by thymidylate synthase
requires N5,N10-Methylene-H4 folate as both one-carbon
source and reducing agent
• N5,N10-Methylene-H4 folate and glycine are produced in
a reversible reaction whereby the hydroxymethyl group
of serine in transferred to H4 folate
• What effect may an elevated glycine:serine ratio during
photorespiration have on DNA synthesis?
January 31, 2008
Catabolism of Purine Nucleotides
Adenosine deaminase deficiency:
• severe immunodeficiency
disease; loss of T- and B-cells
• 100x ↑ dATP (inhibitor of
ribonucleotide reductase)
↓ dNTP’s, ↓ DNA synthesis
Catabolism produces purine bases
for salvage pathways
Uric acid
• catabolic end product in humans
• gout – accumulation of uric acid
in joints and urine
• treatment with xanthine oxidase
inhibitors, e.g. allopurinol
Purine Catabolism
Pyrimidine Catabolism
Salvage Pathways for Nucleotides
• de novo biosynthesis of purine nucleotides assembles
the purine ring on 5’-phosphoribose
• Salvage pathway adds completed purine base to PRPP
– Adenosine phosphoribosyltransferase
Adenine + PRPP → AMP + PPi
– Hypoxanthine-guanine phosphoribosyltransferase
Hypoxanthine + PRPP → IMP + PPi
Guanine + PRPP → GMP + PPi
• Lesch-Nyhan syndrome:
– deficiency in hypoxanthine-guanine phosphoribosyltransferase
Biosynthesis of Cofactors
• Nicotinamide Adenine Dinucleotide (NAD)
PRPP
PPi
Nicotinate
(Niacin)
ATP
PPi
Nicotinate
ribonucleotide
Gln
Glu
Desamido
NAD+
NAD+
• Flavin Adenine Dinucleotide (FAD)
ATP
Riboflavin
ADP
ATP
Riboflavin
5’-phosphate
PPi
FAD