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.
Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia 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 with K(I) and k(inact) values of 5.8 microM 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) and K(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.
Khan
Kishore K
KK
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas 77555-1031, USA. kkkhan@utmb.edu
He
You Qun
YQ
Domanski
Tammy L
TL
Halpert
James R
JR
D019943:Amino Acid Substitution; D018686:Anesthetics, Intravenous / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008874:Midazolam / Q000378:metabolism* / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D051381:Rats; D013223:Statistics as Topic
D018686:Anesthetics, Intravenous; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008874:Midazolam