title: Evaluation of cytochrome P450 mechanism and kinetics using kinetic deuterium isotope effects.
abstract: In this paper two hypotheses are tested: (i) the active oxygen species is similar in energetics for all cytochrome P450 (CYP) enzymes and (ii) linear free-energy relationships can be used to evaluate the mechanism of the reaction of these enzymes. A series of intramolecular isotope effects were determined and compared for CYPs 1A2, 2B1, 2C9, 2E1, and P450cam. The results indicate that the isotope effects are very similar for each of these isoforms of P450 and that the first hypothesis is likely to be true. Attempts to establish a linear free-energy relationship were only moderately successful: log Vmax = 0.11sigma+p + 1.73; r2 = 0.588. It was determined, through the use of intermolecular isotope effects, that the rates of hydrogen atom abstraction are masked. Thus, the second hypothesis is found to be false. This is likely to be a general result for CYP reactions, and linear free-energy relationships can only be used to determine the mechanism under very special circumstances. In all cases, the rate-limiting step should be evaluated with isotope effect experiments before any mechanistic conclusions can be drawn. If the intermolecular isotope effects are found to be masked, no mechanistic conclusion can be drawn from the linear free-energy relationship study.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, New York 14642, USA.
authors.forename: L
authors.initials: L
authors.lastname: Higgins
authors.forename: G A
authors.initials: GA
authors.lastname: Bennett
authors.forename: M
authors.initials: M
authors.lastname: Shimoji
authors.forename: J P
authors.initials: JP
authors.lastname: Jones
mesh_terms: D001667:Binding, Competitive; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry*; D004735:Energy Transfer; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014050:Toluene / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D007527:Isoenzymes; D014050:Toluene; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D019392:Cytochrome P-450 CYP2E1