RT Journal Article SR Electronic T1 Midazolam Oxidation by Cytochrome P450 3A4 and Active-Site Mutants: an Evaluation of Multiple Binding Sites and of the Metabolic Pathway That Leads to Enzyme Inactivation JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 495 OP 506 DO 10.1124/mol.61.3.495 VO 61 IS 3 A1 Kishore K. Khan A1 You Qun He A1 Tammy L. Domanski A1 James R. Halpert YR 2002 UL http://molpharm.aspetjournals.org/content/61/3/495.abstract AB Midazolam (MDZ) oxidation by recombinant CYP3A4 purified fromEscherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding sites. Initial results showed that oxidation of MDZ by CYP3A4 causes time- and concentration-dependent enzyme inactivation withK I and k inactvalues of 5.8 μM and 0.15 min−1, respectively. The different time courses of MDZ hydroxylation by mutants that predominantly formed 1′-OH MDZ as opposed to 4-OH MDZ provided strong evidence that the 1′-OH MDZ pathway leads to CYP3A4 inactivation. Correlational analysis of 1′-OH formation versus 4-OH formation by the mutants supports the inference that the two metabolites result from the binding of MDZ at two separate sites. Thus, substitution of residues Phe-108, Ile-120, Ile-301, Phe-304, and Thr-309 with a larger amino acid caused an increase in the ratio of 1′-OH/4-OH MDZ formation, whereas substitution of residues Ser-119, Ile-120, Leu-210, Phe-304, Ala-305, Tyr-307, and Thr-309 with a smaller amino acid decreased this ratio. Kinetic analyses of nine key mutants revealed that the alteration in regioselectivity is caused by a change in kinetic parameters (V max andK M) for the formation of both metabolites in most cases. The study revealed the role of various active-site residues in the regioselectivity of MDZ oxidation, identified the metabolic pathway that leads to enzyme inactivation, and provided an indication that the two proposed MDZ binding sites in CYP3A4 may be partially overlapping.