Chandrasena R Esala P, Vatsis Kostas P, Coon Minor J, Hollenberg Paul F, Newcomb Martin
Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois 60607, USA.
J Am Chem Soc. 2004 Jan 14;126(1):115-26. doi: 10.1021/ja038237t.
Intramolecular and intermolecular kinetic isotope effects (KIEs) were determined for hydroxylation of the enantiomers of trans-2-(p-trifluoromethylphenyl)cyclopropylmethane (1) by hepatic cytochrome P450 enzymes, P450s 2B1, Delta2B4, Delta2B4 T302A, Delta2E1, and Delta2E1 T303A. Two products from oxidation of the methyl group were obtained, unrearranged trans-2-(p-trifluoromethylphenyl)cyclopropylmethanol (2) and rearranged 1-(p-trifluoromethylphenyl)but-3-en-1-ol (3). In intramolecular KIE studies with dideuteriomethyl substrates (1-d(2)) and in intermolecular KIE studies with mixtures of undeuterated (1-d(0)) and trideuteriomethyl (1-d(3)) substrates, the apparent KIE for product 2 was consistently larger than the apparent KIE for product 3 by a factor of ca. 1.2. Large intramolecular KIEs found with 1-d(2) (k(H)/k(D) = 9-11 at 10 degrees C) were shown not to be complicated by tunneling effects by variable temperature studies with two P450 enzymes. The results require two independent isotope-sensitive processes in the overall hydroxylation reactions that are either competitive or sequential. Intermolecular KIEs were partially masked in all cases and largely masked for some P450s. The intra- and intermolecular KIE results were combined to determine the relative rate constants for the unmasking and hydroxylation reactions, and a qualitative correlation was found for the unmasking reaction and release of hydrogen peroxide from four of the P450 enzymes in the absence of substrate. The results are consistent with the two-oxidants model for P450 (Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555), which postulates that a hydroperoxy-iron species (or a protonated analogue of this species) is a viable electrophilic oxidant in addition to the consensus oxidant, iron-oxo.
测定了反式-2-(对三氟甲基苯基)环丙烷甲烷(1)对映体经肝细胞色素P450酶P450 2B1、Δ2B4、Δ2B4 T302A、Δ2E1和Δ2E1 T303A羟基化反应的分子内和分子间动力学同位素效应(KIEs)。甲基氧化得到两种产物，未重排的反式-2-(对三氟甲基苯基)环丙烷甲醇(2)和重排的1-(对三氟甲基苯基)丁-3-烯-1-醇(3)。在使用双氘代甲基底物(1-d(2))的分子内KIE研究以及使用未氘代(1-d(0))和三氘代甲基(1-d(3))底物混合物的分子间KIE研究中，产物2的表观KIE始终比产物3的表观KIE大1.2左右。通过对两种P450酶进行变温研究表明，在1-d(2)中发现的较大分子内KIE(k(H)/k(D)=10℃时为9-11)不受隧穿效应的影响。结果表明，在整个羟基化反应中需要两个独立的同位素敏感过程，这两个过程要么是竞争性的，要么是连续的。在所有情况下，分子间KIE都被部分掩盖，而对于某些P450酶则大部分被掩盖。结合分子内和分子间KIE结果来确定去掩蔽和羟基化反应的相对速率常数，并且发现在无底物情况下，四种P450酶的去掩蔽反应与过氧化氢释放之间存在定性相关性。这些结果与P450的双氧化剂模型(Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555)一致，该模型假定除了公认的氧化剂铁氧之外，氢过氧铁物种(或该物种的质子化类似物)也是一种可行的亲电氧化剂。
