RESUMO
At least two substitutions were made at each of five amino acid residues in rat cytochrome P450 2B1 that align to residues of known importance in other P450s. The mutants were histidine tagged for purification from Escherichia coli, and the proteins were assessed for testosterone and 7-alkoxycoumarin oxidation. Alteration of each of the sites studied, Phe-115, Ser-294, Phe-297, Ala-298, and Leu-362, was found to affect overall enzyme activity or the metabolite profile. In particular, most of the mutants, excluding F297A, A298G, and L362F, exhibited significantly altered ratios of 16alpha-hydroxytestosterone:16beta-hydroxytestosterone, with the most dramatic alteration being displayed by A298V. Four 7-butoxycoumarin metabolites were produced by CYP2B1, of which two, 7-hydroxycoumarin and 7-(3-hydroxybutoxy)coumarin, were formed at nearly equal rates. Several mutants, F115A, F297A, F297I, and A298V, exhibited an increased predominance of one of the metabolites. The results from this study illustrate the conservation of functionally important residues across P450 subfamilies and families.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Cumarínicos/química , Cumarínicos/metabolismo , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/metabolismo , Esteroides/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/isolamento & purificação , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Modelos Moleculares , Mutação , Oxidantes/metabolismo , Oxirredução , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Esteroide Hidroxilases/química , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/isolamento & purificação , Esteroide Hidroxilases/metabolismo , Esteroides/química , Especificidade por Substrato , Testosterona/química , Testosterona/metabolismoRESUMO
In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B 1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116-134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.
