FCH 532 Lecture 29

Chapter 28: Nucleotide metabolismChapter 24: PhotosynthesisNew study guide posted

Figure 26-1cd Forms of pyridoxal-5-phosphate.(c) Pyridoxamine-5-phosphate (PMP) and (d) The Schiff base that forms between PLP and an enzyme -amino

group.

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Figure 26-13The serine dehydratase reaction.P

age

997

1. Formation of Ser-PLP Schiff base, 2. Removal of the -H atom of serine, 3. elimination of OH-, 4. Hydrolysis of Schiff base, 5. Nonenzymatic tautomerization to the imine, 6. Nonenzymatic hydrolysis to form pyruvate and ammonia.

Serine hydroxymethyltransferase catalyzes PLP-dependent C-C

cleavage

• Catalyzes the conversion of Thr to Gly and acetaldehyde

• Cleaves C-C bond by delocalizing electrons of the resulting carbanion into the conjugated PLP ring:

+N

H

CH3

2-O3PO

CN

HH

O-

H3C-HC--C-COO-

O H

HB:

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Figure 26-54The syntheses of alanine, aspartate, glutamate,

asparagine, and glutamine.

Figure 26-58The conversion of glycolytic intermediate 3-

phosphoglycerate to serine.

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1. Conversion of 3-phosphoglycerate’s 2-OH group to a ketone

2. Transamination of 3-phosphohydroxypyruvate to 3-phosphoserine

3. Hydrolysis of phosphoserine to make Ser.

Purine synthesis

• Purine components are derived from various sources.• First step to making purines is the synthesis of inosine

monophosphate.

De novo biosynthesis of purines: low molecular weight precursors of the purine

ring atoms

Initial derivative is Inosine monophosphate (IMP)

• AMP and GMP are synthesized from IMP

H

P

O-

-O

O Hypoxanthinebase

Inosine monophosphate

Inosine monophosphate (IMP) synthesis

• Pathway has 11 reactions.• Enzyme 1: ribose phosphate pyrophosphokinase • Activates ribose-5-phosphate (R5P; product of pentose phosphate

pathway) to 5-phosphoriobysl--pyrophosphate (PRPP)• PRPP is a precursor for Trp, His, and pyrimidines

• Ribose phosphate pyrophosphokinase regualtion: activated by PPi and 2,3-bisphosphoglycerate, inhibited by ADP and GDP.

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1. Activation of ribose-5-phosphate to PRPP

2. N9 of purine added

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1. Anthranilate synthase

2. Anthranilate phosphoribosyltransferase

3. N-(5’-phosphoribosyl) anthranilate isomerase

4. Indole-3-glycerol phosphate synthase

5. Tryptophan synthase

6. Tryptohan synthase, subunit

7. Chorsmate mutase

8. Prephenate dehydrogenase

9. Aminotransferase

10. Prephenate dehydratase

11. aminotransferase

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1. ATP phosphoribosyltransferase

2. Pyrophosphohydrolase

3. Phosphoribosyl-AMP cyclohydrolase

4. Phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide isomerase

5. Imidazole glycerol phosphate synthase

6. Imidazole glycerol phosphate dehydratase

7. L-histidinol phosphate aminotransferase

8. Histidinol phosphate phosphatase

9. Histidinol dehydrogenase

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Nucleoside diphosphates are synthesized by phosphorylation of nucleoside

monophosphates Nucleoside diphosphates• Reactions catalyzed by nucleoside monophosphate kinases

AMP + ATP 2ADPAdenylate kinase

GMP + ATP GDP + ADPGuanine specific kinase

• Nucleoside monophosphate kinases do not discriminate between ribose and deoxyribose in the substrate (dATP or ATP, for example)

Nucleoside triphosphates are synthesized by phosphorylation of nucleoside monophosphates

Nucleoside diphosphates• Reactions catalyzed by nucleoside diphosphate kinases

ATP + GDP ADP + GTPAdenylate kinase

• Can use any NTP or dNTP or NDP or dNDP

Regulation of purine biosynthesis

• Pathways synthesizing IMP, ATP and GTP are individually regulated in most cells.

• Control total purines and also relative amounts of ATP and GTP.

• IMP pathway regulated at 1st 2 reactions (PRPP and 5-phosphoribosylamine)

• Ribose phosphate pyrophosphokinse- is inhibited by ADP and GDP• Amidophosphoribosyltransferase (1st committed step in the formation of

IMP; reaction 2) is subject to feedback inhibition (ATP, ADP, AMP at one site and GTP, GDP, GMP at the other).

• Amidophosphoribosyltransferase is allosterically activated by PRPP.

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1. Activation of ribose-5-phosphate to PRPP

2. N9 of purine added

Figure 28-5Control network for

the purine biosynthesis

pathway.

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Feedback inhibition is indicated by red arrows

Feedforward activation by green arrows.

Salvage of purines

• Free purines (adenine, guanine, and hypoxanthine) can be reconverted to their corresponding nucleotides through salvage pathways.

• In mammals purines are salvaged by 2 enzymes• Adeninephosphoribosyltransferase (APRT)

Adenine + PRPP AMP + PPi

• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

Hypoxanthine + PRPP IMP + PPi

Guanine + PRPP GMP + PPi

Synthesis of pyrimidines

• Pyrimidines are simpler to synthesize than purines.• N1, C4, C5, C6 are from Asp.• C2 from bicarbonate• N3 from Gln

• Synthesis of uracil monoposphate (UMP) is the first step for producing pyrimidines.

Figure 28-6 The biosynthetic origins of pyrimidine ring atoms.

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Reaction 4: Oxidation of dihydroorateReactions catalyzed by eukaryotic dihydroorotate

dehydrogenase.

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Oxidation of dihydroorotate

• Irreversible oxidation of dihydroorotate to orotate by dihydroroorotate dehydrogenase (DHODH) in eukaryotes.

• In eukaryotes-FMN co-factor, located on inner mitochondrial membrane. Other enzymes for pyrimidine synthesis in cytosol.

• Bacterial dihydroorotate dehydrogenases use NAD linked flavoproteins (FMN, FAD, [2Fe-2S] clusters) and perform the reverse reaction (orotate to dihydroorotate)

Figure 28-9 Reaction 6: Proposed catalytic mechanism for OMP decarboxylase.

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Decarboxylation to form UMP involves OMP decarboxylase (ODCase) to form UMP.

Enhances kcat/KM of decarboxylation by 2 X 1023

No cofactors

Synthesis of UTP and CTP• Synthesis of pyrimidine nucleotide triphosphates is similar to

purine nucleotide triphosphates.• 2 sequential enzymatic reactions catalyzed by nucleoside

monophosphate kinase and nucleoside diphosphate kinase respectively:

UMP + ATP UDP + ADP

UDP + ATP UTP + ADP

Figure 28-10 Synthesis of CTP from UTP.

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CTP is formed by amination of UTP by CTP synthetase

In animals, amino group from Gln

In bacteria, amino group from ammonia

Regulation of pyrimidine nucleotide synthesis

• Bacteria regulated at Reaction 2 (ATCase) • Allosteric activation by ATP• Inhibition by CTP (in E. coli) or UTP (in other bacteria).

• In animals pyrimidine biosynthesis is controled by carbamoyl phosphate synthetase II

• Inhibited by UDP and UTP• Activated by ATP and PRPP• Mammals have a second control at OMP decarboxylase (competitively inhibited by

UMP and CMP)• PRPP also affects rate of OMP production, so, ADP and GDP will inhibit PRPP

production.

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Production of deoxyribose derivatives

• Derived from corresponding ribonucleotides by reduction of the C2’ position.

• Catalyzed by ribonucleotide reductases (RNRs)

ADP dADP

Overview of dNTP biosynthesis

One enzyme, ribonucleotide reductase,reduces all four ribonucleotides to theirdeoxyribose derivatives.

A free radical mechanism is involvedin the ribonucleotide reductasereaction.

There are three classes of ribonucleotidereductase enzymes in nature:Class I: tyrosine radical, uses NDPClass II: adenosylcobalamin. uses NTPs

(cyanobacteria, some bacteria,Euglena).

Class III: SAM and Fe-S to generateradical, uses NTPs.(anaerobes and fac. anaerobes).

Figure 28-12a Class I ribonucleotide reductase from E. coli. (a) A schematic diagram of its

quaternary structure.

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Proposed mechanism for rNDP reductase

Proposed reaction mechanism for ribonucleotide reductase

1. Free radical abstracts H from C3’

2. Acid-catalyzed cleavage of the C2’-OH bond

3. Radical mediates stabilizationof the C2’ cation (unshared electron pair)

4. Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical

5. 3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

Thioredoxin and glutaredoxin

• Final step in the RNR catalytic cycle is the reduction of disulfide bond to reform the redox-active sulfyhydryl pair).

• Thioredoxin-108 residue protein that has redox active Cys (Cys32 and Cys35)-also involved in the Calvin Cycle.

• Reduces oxidized RNR and is regenerated via NADPH by thioredoxin reductase.

• Glutaredoxin is an 85 residue protein that can also reduce RNR.• Oxidized glutaredoxin is reuced by NADPH using glutredeoxin

reductase.

Sources of reducing power for rNDP reductase

Proposed reaction mechanism for ribonucleotide reductase

1. Free radical abstracts H from C3’

2. Acid-catalyzed cleavage of the C2’-OH bond

3. Radical mediates stabilizationof the C2’ cation (unshared electron pair)

4. Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical

5. 3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

dNTPs made by phosphorylation of dNDP

• Reaction is catalyzed by nucleoside diphosphate kinase (same enzyme that phosphorylates NDPs)

dNDP + ATP dNTP + ADP

• Can use any NTP or dNTP as phosphoryl donor.

Thymine synthesis

• 2 main enzymes: dUTP diphosphohydrolase (dUTPase) and thymidylate synthase

Reaction 1• dTMP is made by methylation of dUMP.• dUMP is made by hydrolysis of dUTP via dUTP diphosphohydrolase (dUTPase)

dUTP + H2O dUMP+ PPi

• Done to minimize the concentration of dUTP-prevents incorporation of uracil into DNA.

Thymine synthesis Reaction 2• dTMP is made from dUMP by thymidylate synthase (TS).• Uses N5, N10-methylene-THF as methyl donor

+

+

dUMP

dTMP

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1. Enzyme Cys thiolate group attacks C6 of dUMP (nucleophile).

2. C5 of the enolate ion attacks the CH2 group of the imium cation of N5, N10-methylene-THF.

3. Enzyme base abstracts the acidic proton at C5, forms methylene group and eliminates THF cofactor

4. Migration of the N6-H atom of THF to the exocyclic methylene group to form a methyl group and displace the Cys thiolate intermediate.

Figure 28-19 Catalytic mechanism of thymidylate synthase.

5-flurodeoxyuridylate (FdUMP)

• Antitumor agent.• Irreversible inhibitor of TS• Binds like dUMP but in

step 3 of the reaction, F cannot be extracted.

• Suicide substrate.

FdUMP

F

Figure 28-20The X-ray structure of the E. coli thymidylate synthase–FdUMP–THF ternary complex.

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Thymine synthase oxidizes N5,N10-methyleneTHF

• Only enzyme to change the oxidation state of THF.• Regenerated by 2 reactions• DHF is reduced to THF by NADPH by dihydrofolate

reductase.• Serine hydroxymethyltransferase transfers the

hydroxymethyl group of serine to THF to regenerate N5,N10-methylene-THF and produces glycine.