催化反应的机制mandelate消旋酶:亲电催化谷氨酸317的重要性。
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Mitra B, Kallarakal, Kozarich JW Gerlt是的,克利夫顿詹,Petsko GA,肯扬GL
催化反应的机制mandelate消旋酶:亲电催化谷氨酸317的重要性。
生物化学,1995年3月7日,34 (9):2777 - 87。
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7893689 (在PubMed]
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的高分辨率x射线结构mandelate消旋酶(先生)竞争性抑制剂(S) -atrolactate绑定在活动网站[Landro, j。Gerlt, j。Kozarich, j·W。古,c W。沙,诉J。肯扬,g . L。、Neidhart d J。藤田,J。& Petsko g . a(1994)生物化学33岁,635 - 643年),317年Glu羧酸集团是氢键羧基的抑制剂。这个几何表明317年Glu羧酸官能团一般酸催化剂参与的共同催化一般acid-general基地形成一个稳定的烯醇的互变异构体扁桃酸的反应中间体。为了测试这个假说,先生E317Q突变体的构建和受到高分辨率x射线结构分析(S) -atrolactate的存在。没有观察到陪E317Q替换构象变化在2.1一项决议。的值kcat减少4.5 x 10(3)倍(R) -mandelate和2.9 x 10(4)为(S) -mandelate倍; the values for kcat/Km were reduced 3 x 10(4)-fold. The substrate and solvent deuterium isotope effects measured for both wild-type MR and the E317Q mutant are not multiplicative when deuteriated substrate is studied in D2O, which suggests that the reactions catalyzed by both enzymes are stepwise and involve the formation of stabilized enolic intermediates. In contrast to wild-type MR, E317Q does not catalyze detectable elimination of bromide ion from either enantiomer of p-(bromomethyl)mandelate. However, E317Q is irreversibly inactivated by racemic alpha-phenylglycidate at a rate comparable to that measured for wild-type MR. Taken together, these mechanistic properties confirm the importance of Glu 317 as a general acid catalyst in the reaction catalyzed by wild-type MR. The kcat for wild-type MR and the reduction in kcat observed for E317O are discussed in terms of the analysis recently described by Gerlt and Gassman for understanding the rates and mechanisms of enzyme-catalyzed proton abstraction reactions from carbon acids [Gerlt, J. A., & Gassman, P. G. (1993) J. Am. Chem. Soc. 115, 11552-11568; Gerlt, J. A., & Gassman, P. G. (1993) Biochemistry 32, 11943-11952].