THEORY FOR THE OBSERVED ISOTOPE EFFECTS ON THE FORMATION OF MULTIPLE PRODUCTS BY DIFFERENT KINETIC MECHANISMS OF CYTOCHROME-P450 ENZYMES
THEORY FOR THE OBSERVED ISOTOPE EFFECTS ON THE FORMATION OF MULTIPLE PRODUCTS BY DIFFERENT KINETIC MECHANISMS OF CYTOCHROME-P450 ENZYMES
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DOI：
10.1021/bi00176a024
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发表时间：
1994-03-15
期刊：
影响因子：
2.9
通讯作者：
SUGIYAMA, K
中科院分区：
文献类型：
--
作者：
GILLETTE, JR;DARBYSHIRE, JF;SUGIYAMA, K
细胞色素P450系统是不寻常的，因为它们中的许多可以将底物转化为许多不同的代谢物。可以设想代谢物形成的几种动力学机制。在每种机制中，底物以不同的方向与酶结合形成一组（ES）复合物，然后被激活为一组（EOS）复合物。这些（EOS）配合物的命运决定了动力学机制。在“平行通路”机制中，（EOS）配合物是稳定刚性的，不能直接或间接转化为不同取向的配合物；因此（ES）复合物的取向决定了将形成哪种代谢物。在“非解离”机制中，复合物不是刚性的；相反，当底物停留在酶的活性位点时，它们进行相互转化。在“解离”机制中，（EOS）配合物解离成（EO）和(S)，但重新组合形成具有相同或不同取向的（EOS）配合物。描述这些动力学机制的氘同位素效应的稳态方程已经推导出来，并在孵育混合物中存在相同浓度的氘化和非氘化底物的竞争性实验和只存在一种底物的非竞争性实验中得到了求解。这些方程表明，在两种实验中，通过不涉及从氘化基质中提取氘的途径（非氘提取途径）对代谢物形成的同位素影响的比较可以区分动力学机制。在竞争性实验中，（nu）(H)/(nu)(D)的值为1.0，而在非竞争性实验中，（V-max/K-m）(H)/(V-max/K-m)(D)的值为1.0，这是对“解离”机制的诊断。两种实验值均为1.0为“平行通路”机制的诊断值。的值
Cytochrome P450 systems are unusual in that many of them can convert a substrate to a number of different metabolites. Several kinetic mechanisms may be envisioned by which the metabolites may be formed. In each of the mechanisms, the substrate combines with the enzyme in different orientations to form a set of (ES) complexes that then are activated to a set of (EOS) complexes. The fate of these (EOS) complexes determines the kinetic mechanism. In the ''parallel pathway'' mechanism, the (EOS) complexes are so stable and rigid they cannot be converted either directly or indirectly to complexes with different orientations; the orientation of the (ES) complexes thus determines which metabolite will be formed. In the ''nondissociative'' mechanisms, the complexes are not rigid; instead they undergo interconversion while the substrate remains in the active site of the enzyme. In the ''dissociative'' mechanisms, the (EOS) complexes dissociate to (EO) and (S), but recombine to form (EOS) complexes with either the same or different orientations. Steady-state equations describing the deuterium isotope effects for these kinetic mechanisms have been derived and solved for competitive experiments, in which equal concentrations of both deuterated and nondeuterated substrates are present in incubation mixtures, and for noncompetitive experiments, in which only one of the substrates is present. The equations reveal that comparisons of the isotope effects on the formation of a metabolite by a pathway that does not involve the abstraction of a deuterium from a deuterated substrate (the non-deuterium abstraction pathway) in both experiments can differentiate between the kinetic mechanisms. A value of 1.0 for (nu)(H)/(nu)(D) in the competitive experiment, but 1.0 for the value of (V-max/K-m)(H)/(V-max/K-m)(D) in the noncompetitive experiment, is diagnostic for the ''dissociative'' mechanisms. Values of 1.0 in both kinds of experiments are diagnostic for the ''parallel pathway'' mechanism. Values of