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通过量子力学/分子力学模拟阐明脂肪酸酰胺水解酶及其Lys142Ala变体的水解机制。

Elucidation of hydrolysis mechanisms for fatty acid amide hydrolase and its Lys142Ala variant via QM/MM simulations.

作者信息

Tubert-Brohman Ivan, Acevedo Orlando, Jorgensen William L

机构信息

Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA.

出版信息

J Am Chem Soc. 2006 Dec 27;128(51):16904-13. doi: 10.1021/ja065863s.

Abstract

Fatty acid amide hydrolase (FAAH) is a serine hydrolase that degrades anandamide, an endocannabinoid, and oleamide, a sleep-inducing lipid, and has potential applications as a therapeutic target for neurological disorders. Remarkably, FAAH hydrolyzes amides and esters with similar rates; however, the normal preference for esters reemerges when Lys142 is mutated to alanine. To elucidate the hydrolysis mechanisms and the causes behind this variation of selectivity, mixed quantum and molecular mechanics (QM/MM) calculations were carried out to obtain free-energy profiles for alternative mechanisms for the enzymatic hydrolyses. The methodology features free-energy perturbation calculations in Monte Carlo simulations with PDDG/PM3 as the QM method. For wild-type FAAH, the results support a mechanism, which features proton transfer from Ser217 to Lys142, simultaneous proton transfer from Ser241 to Ser217, and attack of Ser241 on the substrate's carbonyl carbon to yield a tetrahedral intermediate, which subsequently undergoes elimination with simultaneous protonation of the leaving group by a Lys142-Ser217 proton shuttle. For the Lys142Ala mutant, a striking multistep sequence is proposed with simultaneous proton transfer from Ser241 to Ser217, attack of Ser241 on the carbonyl carbon of the substrate, and elimination of the leaving group and its protonation by Ser217. Support comes from the free-energy results, which well reproduce the observation that the Lys142Ala mutation in FAAH decreases the rate of hydrolysis for oleamide significantly more than for methyl oleate.

摘要

脂肪酸酰胺水解酶(FAAH)是一种丝氨酸水解酶,可降解内源性大麻素花生四烯乙醇胺和诱导睡眠的脂质油酰胺,并且作为神经疾病的治疗靶点具有潜在应用价值。值得注意的是,FAAH以相似的速率水解酰胺和酯;然而,当赖氨酸142突变为丙氨酸时,其对酯的正常偏好性又重新出现。为了阐明水解机制以及这种选择性变化背后的原因,我们进行了混合量子力学和分子力学(QM/MM)计算,以获得酶促水解替代机制的自由能分布。该方法的特点是在蒙特卡罗模拟中进行自由能微扰计算,采用PDDG/PM3作为量子力学方法。对于野生型FAAH,结果支持这样一种机制,即质子从丝氨酸217转移到赖氨酸142,同时质子从丝氨酸241转移到丝氨酸217,然后丝氨酸241攻击底物的羰基碳以产生四面体中间体,随后该中间体发生消除反应,同时离去基团通过赖氨酸142 - 丝氨酸217质子穿梭进行质子化。对于赖氨酸142丙氨酸突变体,我们提出了一个引人注目的多步序列,即质子同时从丝氨酸241转移到丝氨酸217,丝氨酸241攻击底物的羰基碳,然后离去基团被消除并由丝氨酸217进行质子化。自由能结果为这一机制提供了支持,这些结果很好地重现了以下观察结果:FAAH中的赖氨酸142丙氨酸突变对油酰胺水解速率的降低幅度明显大于对油酸甲酯水解速率的降低幅度。

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