Role of the alanine at position 363 of cytochrome P450 2B2 in influencing the NADPH- and hydroperoxide-supported activities.

Escherichia coli was used to express the two closely related cytochromes P450 2B1 and 2B2 and two mutants of 2B2 in which residues Gly-303 and Ala-363 were replaced by Ser and Val, respectively. The expressed proteins were partially purified and assayed for benzphetamine and n-octylamine (NOA) binding and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (EOD), benzphetamine N-demethylation (BND) and 7,12-dimethylbenz[a]anthracene (DMBA) hydroxylation activities in the presence and absence of cytochrome b5. The Kd values for benzphetamine and NOA obtained for the wild-type enzymes were similar to reported values. The Ala-363 --> Val mutant (A363V) of 2B2 exhibited Kd values for both ligands that were more similar to 2B1 than to 2B2. The EOD and BND activities of the A363V mutant were 10- and 3.8-fold those exhibited by 2B2, respectively. With DMBA, the A363V mutation led to a 6-fold increase in the hydroxylation activity at the 7-methyl substituent while the hydroxylation activity at the 12-methyl substituent was slightly suppressed. The 7-hydroxymethyl:12-hydroxymethyl product ratio obtained with the A363V mutant (1.3) was much closer to the ratio obtained with 2B1 (1. 9) than to that obtained with 2B2 (0.17). Conversely, the Gly-303 --> Ser substitution did not influence the characteristics of the 2B2-catalyzed metabolism of DMBA to the same magnitude. When cumene hydroperoxide (CHP) was used to support the EOD activities of the proteins, 2B2 exhibited a 2- to 20-fold greater activity than 2B1 or either of the mutants. Examination of the CHP-derived products of the EOD reactions revealed the formation of mainly 2-phenyl-2-propanol due to the heterolytic cleavage of CHP. However, only the 2B1 EOD-reaction mixture also contained the P450-mediated CHP-isomerization products 2-phenyl-1,2-propanediol and 2-(p-hydroxyphenyl)-2-propanol. The formation of these products with 2B1 but not 2B2 may explain why 2B1 is not as efficient as 2B2 or 2B2-G303S in carrying out the CHP-supported reactions.