Structural function of residue-209 in coumarin 7-hydroxylase (P450coh). Enzyme-kinetic studies and site-directed mutagenesis.
Residue-209 plays a critical role in determining the substrate and product specificity of cytochrome P450coh. In order to investigate further the structural function of residue-209 in coumarin 7-hydroxylase reaction, we measured the enzyme-kinetic properties of wild-type P450coh and its mutants in which residue-209 was substituted with various amino acids. In general, the Km and Vmax values for coumarin increased as the size of residue-209 became smaller and Vmax values decreased. The size of residue-209, therefore, was a principle factor determining Km, Kd, and Vmax values of P450coh. Although the polarity and charge also increased the Km value consistently, they altered Vmax and Kd values in an irregular manner. The substitution of serine for residue-209 increased the Vmax, while the substitution of lysine decreased it. Coumarin 7-hydroxylase activity was inhibited weakly by indan, but competitively and strongly by 2-coumaranone. Moreover, Ki values for the inhibitor were similar to Km values of the corresponding, mutated P450s. The results indicate, therefore, that residue-209 is localized in a proposed substrate-binding sequence 1 which binds to the 2-keto group of coumarin and directs its 7-position toward the sixth ligand of heme. Consequently, the identity of residue-209 determines not only the binding of coumarin in P450coh, but also the other reaction step(s) of coumarin 7-hydroxylation.
Juvonen
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Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Iwasaki
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Negishi
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D000596:Amino Acids / Q000737:chemistry; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D000596:Amino Acids; D003374:Coumarins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6