Laughlin L T, Tzeng H F, Lin S, Armstrong R N
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 1998 Mar 3;37(9):2897-904. doi: 10.1021/bi972737f.
Microsomal epoxide hydrolase (MEH) catalyzes the addition of water to epoxides in a two-step reaction involving initial attack of an active site carboxylate on the oxirane to give an ester intermediate followed by hydrolysis of the ester. An efficient bacterial expression system for the enzyme from rat that facilitates the production of native and mutant enzymes for mechanistic analysis is described. Pre-steady-state kinetics of the native enzyme toward glycidyl-4-nitrobenzoates, 1, indicate the rate-limiting step in the reaction is hydrolysis of the alkyl-enzyme intermediate. The enzyme is enantioselective, turning over (2R)-1 about 10-fold more efficiently than (2S)-1, and regiospecific toward both substrates with exclusive attack at the least hindered oxirane carbon. Facile isomerization of the monoglyceride product is observed and complicates the regiochemical analysis. The D226E and D226N mutants of the protein are catalytically inactive, behavior that is consistent with the role of D226 as the active-site nucleophile as suggested by sequence alignments with other alpha/beta-hydrolase fold enzymes. The D226N mutant undergoes hydrolytic autoactivation with a half-life of 9.3 days at 37 degreesC, suggesting that the mutant is still capable of catalyzing the hydrolytic half-reaction (in this instance an amidase reaction) and confirming that D226 is in the active site. The indoylyl side chain of W227, which is in or near the active site, is not required for efficient alkylation of the enzyme or for hydrolysis of the intermediate. However, the W227F mutant does exhibit altered stereoselectivity toward (2R)-1, (2S)-1, and phenanthrene-9,10-oxide, suggesting that modifications at this position might be used to manipulate the stereo- and regioselectivity of the enzyme.
微粒体环氧化物水解酶（MEH）通过两步反应催化水与环氧化物的加成，第一步是活性位点的羧酸盐对环氧乙烷进行初始攻击，生成酯中间体，然后酯发生水解。本文描述了一种高效的大鼠酶细菌表达系统，该系统有助于生产用于机理分析的天然酶和突变酶。天然酶对缩水甘油-4-硝基苯甲酸酯（1）的稳态前动力学表明，反应中的限速步骤是烷基酶中间体的水解。该酶具有对映选择性，对（2R）-1的催化效率比对（2S）-1高约10倍，并且对两种底物都具有区域选择性，优先攻击位阻最小的环氧乙烷碳。观察到甘油单酯产物容易发生异构化，这使区域化学分析变得复杂。该蛋白的D226E和D226N突变体无催化活性，这一行为与序列比对表明D226作为活性位点亲核试剂的作用一致，其他α/β-水解酶折叠酶也有类似情况。D226N突变体在37℃下经历水解自激活，半衰期为9.3天，这表明该突变体仍能够催化水解半反应（在这种情况下是酰胺酶反应），并证实D226位于活性位点。位于活性位点内或附近的W227的吲哚基侧链，对于酶的有效烷基化或中间体的水解不是必需的。然而，W227F突变体对（2R）-1、（2S）-1和菲-9,10-氧化物确实表现出改变的立体选择性，这表明该位置的修饰可用于操纵酶的立体和区域选择性。
