细胞色素P450蛋白酶催化下环己胺脱氨基反应机理的理论研究

Theoreticalinvestigation on the mechanism of the deamination of cyclohexylamine catalyzed by cytochrome P450

在细胞色素P450蛋白酶催化的一系列二级胺和三级胺的脱烷基、脱氨基反应中,环己胺的脱氨基反应有着重要的生理意义,该反应中涉及Cα-H键氧化过程的机理细节在过去几十年中一直存在较大争议,且迄今尚未见相关理论计算报道.针对P450催化的环己胺脱氨基反应,一系列的密度泛函理论(DFT)计算结果及以相关计算数据为基础进行了动力学同位素效应计算结果显示:环己胺底物首先经过一个酶促的Cα-H键氧化反应生成一种甲醇胺,之后又通过一个水分子的辅助在非酶环境中进行了脱氨基反应,生成环己酮和氨气.其中第一步Cα-H键氧化反应涉及了氢原子传递(HAT)过程以及后续无垒的氧反弹过程,且四重态和二重态的能量十分接近,从而证明了Cα-H键氧化反应涉及了"双态反应"(TSR)机理,同时相关的动力学同位素效应计算结果也显示了较大的KIE(动力学同位素效应)值,符合HAT的机理特征.

Plenty of dealkylations or deaminations of both secondary and tertiary amine substrate can be catalyzed by cytochrome P450, among which the deamination of cyclohexylamine is of great physiological meaning. The mechanistic details of the Cα-H bond oxidation process involved in the deamination has been in controversy during the past decades, the corresponding theoretical study has not been reported yet. We carried out a series of density functional theory （DFT） calculations and the kinetic isotope effect （KIE） calculations based on the former calculated results, aiming at the deamination of cyclohexylamine mediated by P450. The results showed that the cyclohexylamine is initially converted to a kind of carbinolamine by enzymatic Cα-H bond oxidation, and then the carbinolamine decomposes to form the product in the non-enzymatic environment assisted by a water molecule. The first step in the Cα-H bond oxidation involves a hydrogen atom transfer process and followed by a barrierless O-rebound process to form the carbinolamine. Besides, the mechanism of the oxidation process is in accord with the two-state reactivity mechanism proposed by Shaik, owing to that the energies of the two spin states are similar. Furthermore, the corresponding calculations of the kinetic isotope effect showed a group of large KIE values which are in agreement with the mechanistic feature of HAT process.