Fig. 5.

Time- and concentration-dependent loss of OHEB carboxylation and determination of the partition ratio for CYP2J2 during inactivation by OD. (A) CYP2J2 in the reconstituted system was incubated with 0 (●), 1 (○), 3 (▴), 20 (Δ), 50 (▪), and 100 μM (□) OD and aliquots were removed at the times indicated and assayed for residual OHEB metabolism as described under Materials and Methods. The catalytic activity at time zero was used as the 100% control to calculate the initial rate constants for the inactivation (k_obs) for each concentration of OD. (B) Shows the fitting of the initial rate constants for the inactivation as a function of the OD concentrations to the Michaelis-Menten equation. The K_I and k_inact values were determined to be 2.5 μM and 0.05 min⁻¹, respectively. (C) Determination of the partition ratio for the inactivation of CYP2J2 by OD. The percentage of catalytic activity remaining was determined as a function of the molar ratio of OD to CYP2J2 as described under Materials and Methods. The partition ratio (∼20) was estimated from the intercept of the linear regression line from the lower ratios of OD to CYP2J2 and the straight line obtained from higher ratios of OD to CYP2J2. The data represent the average of two separate experiments.