Formation of Multiple CYP2E1 Complexes Affects Activity

• Published in: The FASEB journal 2006-03, Vol.20 (4), p.A460-A460
• Main Authors: Jamakhandi, Arvind P, Sanders, Daniel E, Miller, Grover P
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.4.A460

Although there are reports of P450‐P450 interactions between different isoforms on activity, we are the first to report the homotropic effect of CYP2E1 complexation on oxidation of p‐nitrophenol. We performed catalytic titrations of CYP2E1 while varying CPR concentrations and then repeated at different CYP2E1 concentrations to create a family of curves. All titrations were hyperbolic as predicted by a binary complex model, thus we fitted the data globally to a binary mechanism to obtain a proportionality constant of 8.8 ± 0.2 min‐1 and Kd of 26.9 ± 1.1 nM. To assess the effect of complex stoichiometry on catalysis, we measured activity varying CYP2E1 and CPR concentrations, while maintaining a constant total sum of protein. A fit of the data yielded a Kd value of 30 ± 5 nM; however, the curve indicated a maximal activity at ~1:2 CYP2E1:CPR rather than 1:1. Overall, the data indicated that activity was higher than expected when CYP2E1 was limiting and lower when CPR was limiting. To further assess the binary complex model, we performed catalytic titrations of CPR while varying CYP2E1 and repeated the titration at different CPR concentrations to create another family of curves. In contrast to the earlier study, increasing concentrations of CYP2E1 led to an initial increase in activity followed by a significant decrease in activity. Because of the inconsistencies with the binary complex model, we hypothesized that CYP2E1 forms binary and ternary complexes with CPR, and possibly a homodimer. We subjected both sets of titration curves to a multivariable analysis to determine the best mechanism and discuss the implications. These studies were supported by NIH COBRE P20 RR15569.