GLU-320 AND ASP-323 ARE DETERMINANTS OF THE CYP4A1 HYDROXYLATION REGIOSPECIFICITY AND RESISTANCE TO INACTIVATION BY 1-AMINOBENZOTRIAZOLE

Little information is available on the active site structure of the CY P4A family of enzymes or the mechanism by which their omega-hydroxylat ion regiospecificity is enforced. We report here that the E320A, D323E , and E320/D323E mutations decrease the catalytic rate of CYP4A1 simil ar to 5-fold and cause up to a 10-fold shift from omega- to (omega-1)- hydroxylation. The decreased catalytic rate is due to an increase in t he uncoupled reduction of molecular oxygen. Tighter binding of 1- and 4-substituted imidazoles to the double mutant than to the other protei ns suggests that its active site is less constrained. The reaction of these proteins with phenyldiazene causes heme degradation without the detectable formation of a phenyl-iron complex. CYP4A1 and its E320A mu tant are not inactivated by 1-aminobenzotriazole (1-APT), but the D323 E and E320A/D323E mutants are inactivated. The resistance of purified CYP4A 1 to inactivation by I-ABT is surprising in view of the fact tha t 1-ABT causes the loss of the omega-hydroxylase activity both in micr osomal preparations and in vivo. Collectively, the results establish t hat Glu-320, and particularly Asp-323, help to define the active site dimensions, the degree of coupled versus uncoupled turnover, the omega - versus (omega-1)-hydroxylation regiospecificity, and the susceptibil ity to inactivation by mechanism-based inhibitors. Furthermore, they p rovide experimental evidence for a structural analogy between the CYP4 A1 and P450(BM-3) active sites.