Abstract
A mutation at the surface of the substrate access channel which dramatically decreases the affinity for some fatty acids in P450(BM-3) was discovered by random mutagenesis. The mutation introduced, proline-25 to glutamine, is in close proximity to the arginine-47 residue thought to be responsible for the initial docking of fatty acid substrates. The P25Q mutant displays an affinity for palmitate which is approximately 100-fold weaker than the wild-type enzyme. In addition to its altered substrate affinity, P25Q also exhibits altered hydroxylation specificity and carbon monoxide recombination kinetics in the substrate-free form.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Arginine
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Bacterial Proteins*
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Binding Sites
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Cloning, Molecular
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Cytochrome P-450 Enzyme System / chemistry*
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Cytochrome P-450 Enzyme System / metabolism*
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Escherichia coli
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Glutamine
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Kinetics
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Lauric Acids / metabolism
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Mixed Function Oxygenases / chemistry*
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Mixed Function Oxygenases / metabolism*
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Models, Structural
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Mutagenesis, Site-Directed
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Myristic Acid
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Myristic Acids / metabolism
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NADPH-Ferrihemoprotein Reductase
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Palmitic Acid / metabolism
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Point Mutation
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Proline
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Protein Structure, Secondary*
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Substrate Specificity
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Thermodynamics
Substances
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Bacterial Proteins
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Lauric Acids
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Myristic Acids
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Recombinant Proteins
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Myristic Acid
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Glutamine
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lauric acid
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Palmitic Acid
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Cytochrome P-450 Enzyme System
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Arginine
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Proline
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Mixed Function Oxygenases
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NADPH-Ferrihemoprotein Reductase
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flavocytochrome P450 BM3 monoxygenases