Studies on the active site and catalytic mechanism of cytochrome P450 2E1 in heterologous expression systems and microsomes

Cytochrome P450 2E1, the alcohol- and acetone-inducible enzyme, catalyzes the biotransformation of many low-molecular-weight compounds. In order to understand the nature of the P450 2E1 substrate binding site, the competitive inhibition of the P450 2E1-catalyzed N-nitrosodimethylamine demethylase reaction by alcohols and carboxylic acids with varying alkyl chain lengths was studied. With primary alcohols and secondary alcohols, the K{dollar}sb{lcub}rm i{rcub}{dollar} decreased with increasing carbon chain length until the carbon number reached 6 or 7. Among a series of carboxylic acids and {dollar}omega{dollar}-hydroxycarboxylic acids investigated, dodecanoic acid and 12-hydroxydodecanoic acid were the strongest inhibitors, respectively. 1,10-Decanedicarboxylic acid, which contains a carboxylic group at each end, showed a {dollar}rm Ksb{lcub}i{rcub} > 20{dollar} mM. These results suggest that for optimal interaction of a carboxylic acid moiety with the active site of P450 2E1, the hydrocarbon end of the molecule binds to the substrate binding site, leaving the carboxylic acid group outside of a proposed substrate access channel. These results enable me to propose a model to illustrate the structural features of P450 2E1 active site.; Recently, cytochrome P450 BM-3, a bacterial monooxygenase, has been crystallized. In order to compare the catalytic activities of P450 BM-3 and P450 2E1, I studied the metabolism of phenol and benzene, substrates of P450 2E1, by P450 BM-3. These results indicated that both P450 BM-3 and P450 2E1 had similar patterns of phenol metabolism, whereas N-nitrosodimethylamine and p- nitrophenol, typical substrates of P450 2E1, were not substrates for P450 BM-3. These results suggest that P450 2E1 and P450 BM-3 have certain similarities and differences in their catalytic activities.; In order to study the interaction between human P450 2E1 (h2E1) and human P450 reductase (hOR) in a native membrane environment, I developed two approaches to express both h2E1 and hOR in a baculovirus expression system. For dual expression system, the estimated molar expression ratio between hOR and h2E1 in the recombinant membranes was 5:1. The apparent K{dollar}sb{lcub}rm m{rcub}{dollar} and K{dollar}sb{lcub}rm cat{rcub}{dollar} for NDMA demethylase activity were 145 {dollar}mu{dollar}M and 2.4 min{dollar}sp{lcub}-1{rcub}{dollar}, and hb{dollar}sb5{dollar} could cause a 9-fold decrease in K{dollar}sb{lcub}rm m{rcub}{dollar} to 16 {dollar}mu{dollar}M and a 1.6 increase in K{dollar}sb{lcub}rm cat{rcub}{dollar} to 4 min{dollar}sp{lcub}-1{rcub}{dollar}. In a second approach, single expression viruses for each h2E1 and hOR were used to coinfect the Sf9 cells. In this system, I found that h2E1 in the cell membranes decreased with increasing amounts of hOR virus infection. Western and Northern blot analyses of coinfected membranes demonstrate that the suppressing of h2E1 expression by hOR in Sf9 cells is at the protein synthesis level. Stoichiometric analysis with coinfection and triple-infection membranes reveals that hb5 enhances the coupling between h2E1 and hOR for product formation.