5. Wu - Nucleotide Metabolism II

5. Wu - Nucleotide Metabolism II - Conversion of...

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Unformatted text preview: Conversion of nucleotides to deoxyribonucleotides 1 • Replaces 2’-hydroxyl of ribose by hydrogen: • Substrates: NDPs • Ultimate reductant: NADPH Ribonucleotide reductase: Rate limiting step in DNA synthesis 2 Activity of ribonucleotide reductase 3 Identical pairs of subunits B1( α 2) & B2 ( β 2). B1: S –substrate specificity site (high affinity) A-overall activity site (low affinity) Activity control : ATP activates ↑overall activity dATP inhibits↓ overall activity Catalytic site: at the interface between B1 and B2 . cysteine residues : SH groups involved in electron transfer when ribose is reduced to deoxyribose B2 site contains a tyrosine whose OH is crucial to the enzyme activity – target of antitumor agent hydroxyurea Ribonucleotide reductase: the enzyme 4 5 Ribonucleotide reductase •Requires: –thioredoxin –thioredoxin reductase (FAD containing protein) –NADPH • During enzyme catalysis, SH of the cysteine residues oxidized to disulfides -- – Disulfide reduction to free sulfhydryls required to restore ribonucleotide reductase activity • Reduction of disulfides requires a peptide coenzyme present in ribonucleotide reductase, called thioredoxin 6 Ribonucleotide reductase • Oxidation of SH in thioredoxin to disulfides is coupled with restoration of free SH in Cys of ribonucleotide reductase, restoring the enzymatic activity • Oxidized thioredoxin reduced by thioredoxin reductase, coupled with oxidation of NADPH • Ribonucleotide reductase is regulated allosterically by nucleotides 7 Thioredoxin cycle 8 Activity of ribonucleotide reductase 9 S –substrate specificity site (high affinity; allosteric control) A-overall activity site (low affinity allosteric control) binding ATP activates ↑overall activity binding of dATP inhibits ↓ Catalytic site lies at the interface between B1 and B2 . 10 Regulation of ribonucleotide reductase: S site “sense” the relative abundance of dNTPs Also: dATP major –ve effector for all substrates dGTP –ve effector for CDP/UDP Substrate +ve-ve effectors ADP dGTP dATP GDP dTTP dATP dGTP UDP ATP dATP dGTP dTTP CDP ATP dATP dGTP dTTP 11 dUTP pyrophosphatase Thymidylate synthesis 12 One-carbon “charged” THF 13 Thymidylate synthesis/tetrahydrofolate cycle •dUDP → dUTP; enzyme: dUDPkinase •dUTP → dUMP; enzyme: dUTP pyrophosphatase...
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5. Wu - Nucleotide Metabolism II - Conversion of...

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