A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy omega product formation.
A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy omega product formation.
复制标题
确定细胞色素 P450BM3 同一活性位点中两种底物结合的方法：高能 omega 产物形成的解释。
DOI：
10.1016/s0003-9861(03)00228-5
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发表时间：
2003
影响因子：
3.9
通讯作者：
Jones,JeffreyP
中科院分区：
文献类型：
--
作者：
Rock,DanA;Perkins,BrandonNS;Wahlstrom,Jan;Jones,JeffreyP
细胞色素 P450 家族中的许多酶表现出由底物激活、抑制、部分抑制、双相饱和或自动激活引起的非典型（非 Michaelis-Menten）动力学行为。在此，我们提供了一种技术，可以识别由于动力学曲线改变而导致的 P450 同一活性位点中的多个底物占据。利用同位素对产物比率的影响证实，负责棕榈酸（棕榈酸酯）ω 羟基化的酶-底物 (ES) 复合物与导致棕榈酸酯 ω-1 羟基化的 ES 复合物处于快速平衡。第二种底物月桂酸（月桂酸酯）的共孵育会导致 ω 与 ω-1 羟基化棕榈酸酯的比率发生变化。此外，当氘代月桂酸酯与非氘代棕榈酸酯共孵育时，观察到对棕榈酸酯的同位素效应。这些结果仅与两种底物同时位于同一活性位点一致。这种结合模式解释了 P450BM3 的 F87A 突变体如何能够产生 ω 醇，这是一种由高能初级自由基产生的产物。
A number of enzymes from the cytochrome P450 family show atypical (non-Michaelis–Menten) kinetic behavior resulting from substrate activation, inhibition, partial inhibition, biphasic saturation, or autoactivation. Herein, we provide a technique that can identify multiple substrate occupancy in the same active site of a P450 as a result of an altered kinetic profile. Using an isotope effect on product ratios confirms that the enzyme–substrate (ES) complex responsible for ω hydroxylation of palmitic acid (palmitate) is in rapid equilibrium with the ES complex that leads to ω-1 hydroxylation of palmitate. Co-incubation of a second substrate, lauric acid (laurate), results in a change in the ratio of ω to ω-1 hydroxylated palmitate. Furthermore, an isotope effect on palmitate is observed when deuterated laurate is co-incubated with non-deuterated palmitate. These results are only consistent with both substrates being in the same active site simultaneously. This mode of binding explains how the F87A mutant of P450BM3is able to produce the ω alcohol, a product that arises from the high-energy primary radical.