FCH 532 Lecture 28

Chapter 28: Nucleotide metabolismQuiz on Monday essential amino acidsWed. April 11-Exam 3ACS exam is on Monday 5/30Final is scheduled for May 4, 12:45-2:45 PM, in

111 Marshall

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biosynthesis of the “aspartate family” of amino acids: lysine,

methionine, and threonine.

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biosynthesis of the “pyruvate family” of

amino acids: isoleucine, leucine,

and valine.

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Figure 26-62 The biosynthesis of chorismate, the

aromatic amino acid precursor.

Figure 26-63The

biosynthesis of phenylalanine, tryptophan, and

tyrosine from chorismate.

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Figure 26-64A ribbon diagram of the bifunctional enzyme tryptophan synthase from S. typhimurium

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Figure 26-65The biosynthesis of

histidine.

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Hypoxanthine

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.