Derivatives of Amino Acids and Metabolism of Nucleotides
CH353 January 29, 2008
Anabolic Role of the Citric Acid Cycle
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
Purines
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
bacteria, plants
heme precursor
Biosynthesis of Heme
Genetic Deficiencies in Heme Biosynthesis
Catabolism of Heme
Regulated step: 3 isozymes
Important serum antioxidant
Bile pigment
purple
green
yellow
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 reactionsL-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 reactionsHistidine → 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
R–C–O–
O
C–C–R
O
H
ATP ADP R–C–OPO3–2
O
C–C–R
OPO3–2
R’NH2 PO4–2 R–C–NHR’
O
C–C–R
NHR’
Biosynthesis of the Purine Ring
• Multi-step synthesis from many precursors– (numbers indicate order of addition to purine ring from PRPP)
1
2
3
4
5
6
7
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 HCO3
-
• carbamoylation 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• Hypoxanthine (a purine) is
assembled on the ribose 5-phosphate → Inosinate (IMP)
• Precursors:– PRPP– Glycine– H4 folate-formate (2)
– HCO3–
– Glutamine (amide-N) (2)– Aspartate (amino-N)
• IMP → AMP• IMP → XMP → GMP
Pyrimidine Biosynthesis• Orotate (a pyrimidine) is made
first then added to ribose 5-phosphate → 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 carbamoyl phosphate synthetase II
A aspartate transcarbamoylase
D 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 + NTPdonor 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 ReductaseBinding at activity regulatory sites:• ATP activates enzyme
• dATP inhibits enzyme
Binding at substrate specificity sites:• 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
UTP
nucleoside diphosphate
kinase
UMP
cytidylate synthetase
uridylate kinase
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)
Nicotinate (Niacin)
PRPP PPi
Nicotinate ribonucleotide
ATP PPi
Desamido NAD+
Gln Glu
NAD+
• Flavin Adenine Dinucleotide (FAD)
RiboflavinRiboflavin
5’-phosphateFAD
ADPATP PPiATP