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  • Title: Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants.
    Author: Kobayashi Y, Fang X, Szklarz GD, Halpert JR.
    Journal: Biochemistry; 1998 May 12; 37(19):6679-88. PubMed ID: 9578551.
    Abstract:
    A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order of C2 > C4 > C3 > C1 > C5 > C6 = C7. The pattern observed for the P450 I114V mutant was similar to that of the wild-type enzyme, whereas with F206L and G478S mutants, the rate of O-dealkylation was low with all the compounds. In contrast, with V363A, the highest rate of product formation was observed with 7-butoxycoumarin. The V363L mutant preferentially catalyzed the O-dealkylation of 7-methoxy- and 7-ethoxycoumarin, and a further increase in the length of the alkyl chain led to a marked decrease in product formation. The stoichiometry of 7-butoxycoumarin oxidation by V363L suggested that products other than 7-hydroxycoumarin were also formed. HPLC and GC-EIMS analyses revealed that P450 2B1 V363L produced 7-(3-hydroxybutoxy)coumarin and 7-(4-hydroxybutoxy)coumarin as major oxidation products, while the V363A mutant mainly catalyzed the O-dealkylation of 7-butoxycoumarin. Docking of alkoxycoumarins into the active site of a P450 2B1 homology model confirmed the importance of the studied residues in substrate dealkylation and explained the formation of novel 7-butoxycoumarin products by the V363L mutant.
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