Rock Dan A, Perkins Brandon N S, Wahlstrom Jan, Jones Jeffrey P
Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
Arch Biochem Biophys. 2003 Aug 1;416(1):9-16. doi: 10.1016/s0003-9861(03)00228-5.
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 omega hydroxylation of palmitic acid (palmitate) is in rapid equilibrium with the ES complex that leads to omega-1 hydroxylation of palmitate. Co-incubation of a second substrate, lauric acid (laurate), results in a change in the ratio of omega to omega-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 P450BM3 is able to produce the omega alcohol, a product that arises from the high-energy primary radical.
细胞色素P450家族的许多酶表现出非典型（非米氏）动力学行为，这是由底物激活、抑制、部分抑制、双相饱和或自激活引起的。在此，我们提供了一种技术，该技术可通过改变的动力学曲线识别P450同一活性位点中的多种底物占据情况。利用对产物比例的同位素效应证实，负责棕榈酸（棕榈酸盐）ω-羟基化的酶-底物（ES）复合物与导致棕榈酸盐ω-1羟基化的ES复合物处于快速平衡状态。第二种底物月桂酸（月桂酸盐）的共同孵育导致ω-羟基化与ω-1羟基化棕榈酸盐比例发生变化。此外，当氘代月桂酸盐与非氘代棕榈酸盐共同孵育时，观察到对棕榈酸盐的同位素效应。这些结果仅与两种底物同时存在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。
