Derivatives of Amino Acids and Metabolism of Nucleotides
description
Transcript of Derivatives of Amino Acids and Metabolism of Nucleotides
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