Probing the Active Sites of Rat and human cytochrome-P450 2E1 with alcohols and carboxylic acids

Cytochrome P450 2E1 (P450 2E1) catalyzes the biotransformation of many low-molecular-weight compounds including industrial solvents, indoor pollutants, alcohol, and drugs. In order to understand the nature of the P450 2E1 active site, we studied the competitive inhibition of the P450 2E1-catalyzed N-nitrosodimethylamine demethylation by alcohols and carboxylic acids with different alkyl chain lengths. Using microsomes from acetone-treated rats as the enzyme source of P450 2E1, the K_i value for each compound was measured. With primary alcohols and secondary alcohols, the K_i decreased with the increase in the carbon chain length until the carbon number reached 6 or 7; the free energy increment of binding was 0.28 kcal/mol CH₂ group. Similar inhibitory effects were also observed with human P450 2E1 heterologously expressed in Hep G2 cells. These results suggest that both rat and human P450 2E1 contain a pocket with hydrophobicity that serves as a binding site for low-molecular-weight substrates. Among a series of carboxylic acids and ω-hydroxycarboxylic acids investigated, dodecanoic acid was the strongest inhibitor (K_i = 22 μM), and 12-hydroxydodecanoic acid had the lowest K_i (320 μM) within the series of ω-hydroxycarboxylic acids. The free energy increment of binding for carboxylic acids was 0.35 kcal/mol CH2 group. 1, 10-Decanedicarboxylic acid, which contains a carboxylic group at each end, showed a K_i > 20 mM. We 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. The length of optimal substrates such as dodecanoic acid may reflect the length of the substrate access channel and the size of the active site. We estimate that the distance from the opening of the access channel to the oxygenation site is about 15 Å. Based on the structural features of P450 2E1 substrates and competitive inhibitors, we propose a conceptual model to illustrate the binding of these molecules in the active site of P450 2E1.