Sheng Xin, Zhang Haoming, Hollenberg Paul F, Newcomb Martin
Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Biochemistry. 2009 Feb 24;48(7):1620-7. doi: 10.1021/bi802279d.
Kinetic isotope effects were measured for oxidations of (S,S)-2-(p-trifluoromethylphenyl)cyclopropylmethane containing zero, two, and three deuterium atoms on the methyl group by Compounds I from the cytochrome P450 enzymes CYP119 and CYP2B4 at 22 degrees C. The oxidations displayed saturation kinetics, which permitted solution of both binding constants (K(bind)) and first-order oxidation rate constants (k(ox)) for both enzymes with the three substrates. The binding constant for CYP2B4 Compound I was about 1 order of magnitude greater than that for CYP119 Compound I, but the oxidation rate constants were similar for the two. In oxidations of 1-d(0), k(ox) = 10.4 s(-1) for CYP119 Compound I, and k(ox) = 12.4 s(-1) for CYP2B4 Compound I. Primary kinetic isotope effects (P) and secondary kinetic isotope effects (S) were obtained from the results with the three isotopomers. The primary KIEs were large, P = 9.8 and P = 8.9 for CYP119 and CYP2B4 Compounds I, respectively, and the secondary KIEs were small and normal, S = 1.07 and S = 1.05, respectively. Large intermolecular KIEs for 1-d(0) and 1-d(3) of k(H)/k(D) = 11.2 and 9.8 found for the two Compounds I contrast with small intermolecular KIEs obtained previously for the same substrate in P450-catalyzed oxidations; these differences suggest that a second electrophilic oxidant, presumably iron-complexed hydrogen peroxide, is important in cytochrome P450 oxidations under turnover conditions.
在22摄氏度下，测量了细胞色素P450酶CYP119和CYP2B4的化合物I对甲基上含有零个、两个和三个氘原子的(S,S)-2-(对三氟甲基苯基)环丙基甲烷的氧化反应的动力学同位素效应。氧化反应呈现出饱和动力学，这使得能够求解两种酶与三种底物的结合常数（K(bind)）和一级氧化速率常数（k(ox)）。CYP2B4化合物I的结合常数比CYP119化合物I的大约大1个数量级，但两种酶的氧化速率常数相似。在1-d(0)的氧化反应中，CYP119化合物I的k(ox) = 10.4 s(-1)，CYP2B4化合物I的k(ox) = 12.4 s(-1)。从三种同位素异构体的结果中获得了一级动力学同位素效应（P）和二级动力学同位素效应（S）。一级动力学同位素效应较大，CYP119和CYP2B4化合物I的P分别为9.8和8.9，二级动力学同位素效应较小且正常，分别为S = 1.07和S = 1.05。两种化合物I的1-d(0)和1-d(3)的分子间动力学同位素效应k(H)/k(D)分别为11.2和9.8，与之前在P450催化氧化中对相同底物获得的小分子间动力学同位素效应形成对比；这些差异表明，第二种亲电氧化剂，可能是铁络合的过氧化氢，在周转条件下的细胞色素P450氧化中很重要。
