Sequence alignment of human cytochrome P450 3A4 with bacterial enzymes of known structure has provided a basis from which to predict residues involved in substrate oxidation. Substitutions were made at four residues (I301, F304, A305, and T309) predicted to be located within the highly conserved substrate recognition site 4. Site-directed mutants engineered to contain carboxy-terminal histidine tags were expressed in Escherichia coli and purified on a metal affinity column. The integrity of each protein was assessed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Functional analysis was performed using progesterone and testosterone as substrates and alpha-naphthoflavone as an activator. In testosterone hydroxylase assays, all of the mutants displayed rates of total product formation similar to wild-type 3A4, with several mutants showing small differences in specific products formed. However, with progesterone as the substrate, mutants F304A, A305V, and T309A exhibited altered product ratios and/or changes in the rates of product formation. F304A and A305V also displayed altered flavonoid stimulation that resulted in product ratios dramatically different from wild-type 3A4. Therefore, the kinetics of progesterone hydroxylation of these mutants and the wild-type enzyme were further assessed, and the data were analyzed with the Hill equation. Results with wild-type 3A4 and F304A indicated that at high progesterone concentrations, hydroxylation rates and product ratios are independent of the presence of alpha-NF. This suggests that progesterone may be equivalent to alpha-NF as an activator. In contrast, A305V exhibited autoactivation by progesterone but inhibition by alpha-NF.
Copyright 1998 Academic Press.