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Vol. 61, Issue 3, 495-506, March 2002
Department of Pharmacology and Toxicology, The University of Texas
Medical Branch, Galveston, Texas.
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
KI and kinact
values 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 (Vmax and
KM) 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.
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