PT  - JOURNAL ARTICLE
AU  - Domanski, Tammy L.
AU  - He, You-Ai
AU  - Harlow, Greg R.
AU  - Halpert, James R.
TI  - Dual Role of Human Cytochrome P450 3A4 Residue Phe-304 in Substrate Specificity and Cooperativity
DP  - 2000 May 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 585--591
VI  - 293
IP  - 2
4099  - http://jpet.aspetjournals.org/content/293/2/585.short
4100  - http://jpet.aspetjournals.org/content/293/2/585.full
SO  - J Pharmacol Exp Ther2000 May 01; 293
AB  - 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 α-naphthoflavone, exhibiting a 2-fold decrease in theKm value for progesterone 6β-hydroxylation in the presence of 25 μM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained theVmax and decreased theKm for progesterone 6β-hydroxylation, minimized stimulation by α-naphthoflavone, and decreased the rate of α-naphthoflavone oxidation to one-eighth of the wild type. Interestingly, the ΔAmax for spectral binding of α-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and α-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen. The American Society for Pharmacology and Experimental Therapeutics