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Vol. 293, Issue 2, 585-591, May 2000
University of Texas Medical Branch, Department of Pharmacology and
Toxicology, Galveston, Texas
The structural basis of cooperativity of progesterone hydroxylation
catalyzed by human cytochrome P450 3A4 has been investigated. A recent
study suggested that substitution of larger side chains at positions
Leu-211 and Asp-214 partially mimics the action of effector by reducing
the size of the active site. Based on predictions from molecular
modeling that Phe-304 in the highly conserved I helix is involved in
both effector and substrate binding, a tryptophan residue was
substituted at this position. The purified F304W mutant displayed
hyperbolic progesterone hydroxylase kinetics, indicating a lack of
homotropic cooperativity. However, the mutant remained responsive to
stimulation by 
-naphthoflavone, exhibiting a 2-fold decrease in the
Km value for progesterone 6
-hydroxylation
in the presence of 25 µM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained the
Vmax and decreased the
Km for progesterone 6-hydroxylation,
minimized stimulation by -naphthoflavone, and decreased the rate of
-naphthoflavone oxidation to one-eighth of the wild type.
Interestingly, the 
Amax for spectral
binding of -naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and -naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket,
although both substrates appear to have equal access to the reactive oxygen.
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