Steady-state kinetics of oxidation of ethanol by human liver cytochrome P450 2E1 (CYP 2E1) in a liposomal reconstituted system

The microsomal ethanol oxidizing system (MEOS) is embedded in the endoplasmic reticulum of the eukaryotic cells. The major components of MEOS, cytochrome P450 2E1 (CYP 2E1) and a redox partner NADPH-cytochrome P450 oxidoreductase (OR), are associated in the form a multi-enzyme complex that oxidizes ethanol. The measurement of ethanol oxidation by CYP 2E1 within a reconstituted system requires the presence of 1,2-dilauroyl-sn-glycero-3-phosphocholine (12:0) and cytochrome b₅ (cyt b₅) in addition to OR, for optimal CYP 2E1 catalytic activity. The goal of this research was to develop a comprehensive rate equation that describes the overall rate of oxidation of ethanol by CYP 2E1 in a liposomal reconstituted system.; In the preliminary studies, initial rates of oxidation of ethanol by CYP 2E1 in microsomal system were measured at several concentrations of ethanol and NADPH, in the presence of phospholipids (12:0) and (18:0) separately. Two non-linear regression models, mech 1 and mech 3 based on variations of ordered Bi-Bi mechanism, were used to fit initial rate data and steady-state kinetic constants were determined. The variation in phospholipid chain length from (12:0) to (18:0) or concentration of phospholipid (12:0) had no effect on the steady-state kinetics.; Methods were developed to incorporate purified CYP 2E1, cyt b₅ and OR into phospholipid (12:0) in order to create a liposomal reconstituted system. Initial rates of oxidation of ethanol were measured at several concentrations of substrates ethanol and NADPH, enzymes OR and cyt b₅. In addition, new non-linear regression kinetic models were derived to examine the effects of concentration of substrates, enzymes and total phosphorus (from phospholipid (12:0)) on the steady-state kinetics. The initial rate data were analyzed using rate equations based on these models in SAS (Cary, NC). It was determined that increase in concentration of OR, cyt b₅ and total phosphorus decreases K_mETOH and K_mNADPH without affecting V_max. A comprehensive rate equation describing the overall rate of oxidation of ethanol by CYP 2E1 was developed. The results provide additional insight into essential role of OR, cyt b₅ and total phosphorus in the binding affinity of substrates to CYP 2E1-OR complex.