FCH 532 Lecture 29

Chapter 28: Nucleotide metabolismQuiz on Mon (4/16): IMP synthesis-Purine synthesisQuiz on Wed(4/18): Pyrimidine biosynthesis/regulationQuiz on Friday(4/20): Ribonucleotide reductase

mechanismFriday (4/20): extra credit seminar, Dr. Jimmy Hougland,145 Baker, 3-4PM.ACS exam has been moved to Monday (4/30)Quiz on Final is scheduled for May 4, 12:45PM-2:45PM,

in 111 Marshall

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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.

Figure 28-21Regeneration of N5,N10-methylenetetrahydrofolate.

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Nucleotide degradation• Nucleic acids can survive the acid of the stomach • Degraded into nucleotides by pancreatic nucleases and intestinal

phosphodiesterases in the duodenum.• Components cannot pass through cell membranes, so they are hydrolyzed to

nucleosides.• Nucleosides may be directly absorbed by the intestine or undergo further

degradation to free bases and ribose or ribose-1-phosphate by nucleosidases and nucloside phosphorylase.

Nucleoside + H2O base + ribose

Nucleoside + Pi base + ribose-1-PNucleoside phosphorylase

nucleosidase

Catabolism of purines• All pathways lead to formation of uric acid.• Intermediates could be intercepted into salvage pathways.• 1st reaction is the nucleotidase and second is catalyzed by purine nucleoside

phosphorylase (PNP)

• Ribose-1-phosphate is isomerized by phosphoribomutase to ribose-5-phosphate (precursor to PRPP).

Purine nucleoside + Pi Purine base + ribose-1-P

• Adenosine and deoxyadenosine are not degraded by PNP but are deaminated by adenosine deaminase (ADA) and AMP deaminase in mammals

Purine nucleoside phosphorylase

Figure 28-23Major pathways of purine catabolism in animals.

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ADA

Genetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese (SCID).

No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPs

Figure 28-24a Structure and mechanism of adenosine deaminase. (a) A ribbon diagram of murine

adenosine deaminase in complex with its transition state analog HDPR.

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Figure 28-24b (b) The proposed catalytic mechanism of adenosine deaminase.

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1. Zn2+ polarized H2O molecule nucleophilically attacks C6 of the adenosine. His is general base catalyst, Glu is general acid, and Asp orients water.

2. Results in tetrahedral intermediate which decomposes by elimination of ammonia.

3. Product is inosine in enol form (assumes dominant keto form upon release from enzyme).

Purine nucleotide cycle• Deamination of AMP to IMP combined with synthesis of AMP

from IMP results in deaminating Asp to yield fumarate.• Important role in skeletal muscle-increased activity requires

increased activity in the citric acid cycle.• Muscle replenishes citric acid cycle intermediates through the

purine nucleotide cycle.

Figure 28-25The purine nucleotide cycle.

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Xanthine oxidase• Xanthine oxidse (XO) converts hypoxanthine to xanthine, and xanthine

to uric acid.• In mammals, found in the liver and small intestine mucosa• XO is a homodimer with FAD, two [2Fe-2S] clusters and a molybdopterin

complex (Mo-pt) that cycles between Mol (VI) and Mol (IV) oxidation states.

• Final electron acceptor is O2 which is converted to H2O2

• XO is cleaved into 3 segments. The uncleaved enzyme is known as xanthine dehydrogenase (uses NAD+ as an electron acceptor where XO does not).

• XO hydroxylates hypoxanthine at its C2 position and xanthine at the C8 positon to produce uric acid in the enol form.

Figure 28-26a X-Ray structure of xanthine oxidase from cow’s milk in complex with salicylic acid.

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N-terminal domain is cyan

Central domain is gold

C-terminal domain is lavender

Mechanism for XO1. Reaction initiated by attack of enzyme nucleophile on the C8

position of xanthine.

2. The C8-H atom is eliminated as a hydride ion that combines with Mo (VI) complex, reducing it to Mo (IV).

3. Water displaces the enzyme nucleophile producing uric acid.

Figure 28-27Mechanism of xanthine oxidase.

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Figure 28-23Major pathways of purine catabolism in animals.

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ADA

Genetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese (SCID).

No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPs

Purine degredation in other animals

Primates, birds, reptiles, insects-final degradation product id uric acid which is excreted in urine.

Goal is the conservation of water.

Figure 28-29The Gout, a cartoon by James Gilroy (1799).

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Gout is a disease characterized by elevated levels of uric acid in body fluids. Caused by deposition of nearly insoluble crystals of sodium urate or uric acid.

Clinical disorders of purine metabolism

Excessive accumulation of uric acid: Gout

The three defects shown each result in elevated de novo purine biosynthesis

Common treatment for gout: allopurinol

Allopurinol is an analogue of hypoxanthine that strongly inhibits xanthine oxidase. Xanthine and hypoxanthine, which are soluble, are accumulated and excreted.

Catabolism of pyrimidines

• Animal cells degrade pyrimidines to their component bases.• Happen through dephosphorylation, deamination, and

glycosidic bond cleavage.• Uracil and thymine broken down by reduction (vs. oxidation

in purine catabolism).

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Biosynthesis of of NAD and NADP+

Produced from vitamin precursors Nicotinate and Nicotinamide and from quinolinate, a Trp degradation product

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Biosynthesis of FMN and FAD from riboflavin

FAD is synthesized from riboflavin in a two-reaction pathway.

Flavokinase phosphorylates the 5’OH group to give FMN

FAD pyrophosphorylase catalyzes the next step (coupling of FMN to ADP).

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Biosynthesis of CoA from pantothenate