Glu-320 and Asp-323 Are Determinants of the CYP4A1 Hydroxylation Regiospecificity and Resistance to Inactivation by 1-Aminobenzotriazole

Abstract

Little information is available on the active site structure of the CYP4A family of enzymes or the mechanism by which their omega-hydroxylation regiospecificity is enforced. We report here that the E320A, D323E, and E320/D323E mutations decrease the catalytic rate of CYP4A1 approximately 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 the uncoupled reduction of molecular oxygen. Tighter binding of 1- and 4-substituted imidazoles to the double mutant than to the other proteins 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 mutant are not inactivated by 1-aminobenzotriazole (1-ABT), but the D323E and E320A/D323E mutants are inactivated. The resistance of purified CYP4A1 to inactivation by 1-ABT is surprising in view of the fact that 1-ABT causes the loss of the omega-hydroxylase activity both in microsomal preparations and in vivo. Collectively, the results establish that Glu-320, and particularly Asp-323, help to define the active site dimensions, the degree of coupled versus uncoupled versus uncoupled turnover, the omega-versus (omega-1)-hydroxylation regiospecificity, and the susceptibility to inactivation by mechanism-based inhibitors. Furthermore, they provide experimental evidence for a structural analogy between the CYP4A1 and P450BM-3 active sites.

Calibration of the Channel That Determines the ω-Hydroxylation Regiospecificity of Cytochrome P4504A1

“…2, inset). These measurements gave the following values for the dissociation constants of the three halododecanoic acids: 12-chloro, K s ϭ 11.8 M; 12-bromo, K s ϭ 7.9 M; and 12-iodo, K s ϭ 6.0 M. Comparison of these values with K s ϭ ϳ30 M for dodecanoic acid itself indicates that all three 12-halododecanoic acids bind to CYP4A1 with similar affinities but 3-4-fold more tightly than the parent dodecanoic acid (20).…”

“…MO). CYP4A1 was expressed and purified as previously reported (20). Human CYP2E1 (ϩ P450 reductase ϩ cytochrome b 5 Supersomes TM ) and control insect cell membranes were purchased from Gentest-BD Biosciences.…”

“…Human CYP2E1 (ϩ P450 reductase ϩ cytochrome b 5 Supersomes TM ) and control insect cell membranes were purchased from Gentest-BD Biosciences. The human NADPH-cytochrome P450 reductase cDNA was provided by Stephen M. Black and the protein was expressed and purified as previously described (20). Purified rat liver cytochrome b 5 was a gift from Lester Bornheim (University of California, San Francisco).…”

Repeated whiskey binges promote liver injury in rats fed a choline-deficient diet

“…Oxygen radicals modify proteins leading to their inactivation and rapid degradation (21). Among the various oxidative modifications of aminoacids, carbonyl formation may be an early marker for protein oxidation (34). Oxidative stress occurred especially in rats fed the CD diet plus repeated whiskey binges.…”

Genetics of P450 oxidoreductase: Sequence variation in 842 individuals of four ethnicities and activities of 15 missense mutations

“…Therefore, we supplemented the cytochrome c reduction/NADPH oxidation assays with assays that directly measure the capacity of POR to support the activities of P450c17. These assays combined membranes containing Nterminally modified full-length human P450c17 expressed in bacteria (21) and a membrane fraction containing human N-27 POR mutants expressed in Escherichia coli (22). Cytochrome b 5 , which acts as an allosteric factor to facilitate the 17,20 lyase activity of P450c17 (23), was added to support the lyase reaction, and the K m , V max , and V max /K m for the 17␣-hydroxylase and 17,20 lyase activities of P450c17 were determined by Lineweaver-Burke analysis ( Table 3).…”