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