FCH 532 Lecture 28 Chapter 28: Nucleotide metabolism Quiz on Monday essential amino acids Wed. April...
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Transcript of FCH 532 Lecture 28 Chapter 28: Nucleotide metabolism Quiz on Monday essential amino acids Wed. April...
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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39Figure 26-60The
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.