STEREOCHEMISTRY AND DEUTERIUM-ISOTOPE EFFECTS IN CAMPHOR HYDROXYLATION BY THE CYTOCHROME P450CAM MONOXYGENASE SYSTEM
STEREOCHEMISTRY AND DEUTERIUM-ISOTOPE EFFECTS IN CAMPHOR HYDROXYLATION BY THE CYTOCHROME P450CAM MONOXYGENASE SYSTEM
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DOI：
10.1021/bi00531a026
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
SLIGAR, SG
中科院分区：
文献类型：
--
作者：
GELB, MH;HEIMBROOK, DC;SLIGAR, SG
细菌(恶臭假单胞菌)细胞色素P450cam在体内催化樟脑羟化生成5-外羟基樟脑，并在O2、吡啶核苷酸、黄素蛋白脱氢酶和腐胺还蛋白重组体系中催化生成5-外羟基樟脑。当铁形态的血球蛋白与外源氧化剂碘苯、间氯苯酸和过氧化氢混合时，也会形成产物。当研究P450cam依赖的羟基化反应时，在5-exo或5-endo位置上的樟脑类似物，观察到非常小的分子间同位素对整个反应速度的影响，并记录了显著的分子内同位素效应。提出了中间底物Ca自由基的存在。明显地，从樟脑骨架上C-5的Exo或Endo位置可以发生抽提，O2只立体地添加到表面，得到唯一的产物5-exo-羟基樟脑。使用这些底物，对于吡啶核苷酸/O2和外源氧化剂，产物乙醇中几乎相同的H/H同位素比率支持羟化反应，表明这些反应共享一个共同的抽氢物种。测量到的相对较小的分子内同位素效应可以用一个包含可逆氢抽提步骤和/或重原子在反应坐标中运动的模型来合理化。
Bacterial (Pseudomonas putida) cytochrome P450cam catalyzes the hydroxylation of camphor to yield 5-exo-hydroxycamphor in vivo and in a reconstituted system with O2, pyridine nucleotide, flavoprotein dehydrogenase and putidaredoxin. Product is also formed when the ferric form of the hemoprotein is mixed with the exogenous oxidants iodosobenzene, m-chloroperbenzoic acid and H2O2. When the P450cam-dependent hydroxylation reactions are studied with camphor analogs containing deuterium at either the 5-exo or 5-endo position, a very small intermolecular isotope on the overall reaction velocity is observed and a significant intramolecular isotope effect is documented. The existence of an intermediate substrate Ca radical is suggested. Abstraction can apparently occur from either the exo or endo position at C-5 on the camphor skeleton, with the O2 stereospecifically added to only the Re face to give 5-exo-hydroxycamphor as the unique product. Using these substrates, nearly identical H/deuterium isotope ratios in the product alcohol for the pyridine nucleotide/O2 and exogenous oxidant supported hydroxylations, suggesting that these reactions share a common H-abstracting species. The relatively small magnitude of the measured intramolecular isotope effect can be rationalized with a model involving a reversible H-abstraction step and/or the involvement of heavy-atom motion in the reaction coordinate.