Abstract
The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme. The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation.
Copyright 2000 Academic Press.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Anti-HIV Agents / metabolism*
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Binding Sites
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Catalysis
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Crystallography, X-Ray
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Dideoxynucleotides
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Enzyme Stability
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Humans
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Kinetics
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Models, Molecular
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Mutation / genetics*
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Nucleoside-Phosphate Kinase / chemistry*
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Nucleoside-Phosphate Kinase / genetics
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Nucleoside-Phosphate Kinase / metabolism*
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Nucleotides / metabolism
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Phosphorylation
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Prodrugs / metabolism*
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Protein Binding
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Protein Conformation
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Thymine Nucleotides / metabolism*
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Zidovudine / analogs & derivatives*
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Zidovudine / metabolism*
Substances
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Anti-HIV Agents
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Dideoxynucleotides
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Nucleotides
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Prodrugs
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Recombinant Fusion Proteins
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Thymine Nucleotides
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3'-azido-3'-deoxythymidine 5'phosphate
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Zidovudine
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Nucleoside-Phosphate Kinase
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dTMP kinase