Poyner R R, Laughlin L T, Sowa G A, Reed G H
Institute for Enzyme Research, Graduate School, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53705, USA.
Biochemistry. 1996 Feb 6;35(5):1692-9. doi: 10.1021/bi952186y.
High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q >> E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction.
高分辨率晶体学数据表明，谷氨酸168（Glu 168）和谷氨酸211（Glu 211）位于活性位点与赖氨酸345（Lys 345）相对的表面。这些结构研究产生了两种关于一般碱催化的不同提议。在一种方案中，有人提出谷氨酸168和谷氨酸211的羧酸盐侧链使捕获的水分子电离，产生的OH⁻充当碱[莱比奥达，L.，& 斯特克，B.（1991年）《生物化学》30卷，2817 - 2822页]。在另一种提议中，赖氨酸345的ε - 氨基在一般碱催化中发挥作用[韦德金德，J. E.，波伊纳，R. R.，里德，G. H.，& 雷门特，I.（1994年）《生物化学》33卷，9333 - 9342页]。已经制备了编码酵母烯醇化酶这些活性位点残基的位点特异性突变体的基因，即K345A、E168Q和E211Q。野生型和突变基因各自的蛋白质产物在大肠杆菌中表达并以均一形式分离出来。所有三种突变蛋白在整体反应中的活性都严重降低——不到野生型活性的1/10⁵。研究了三种突变蛋白在部分反应中的性质，以更清楚地确定这些残基在催化循环中的作用。K345A变体无法催化2 - 磷酸 - D - 甘油酸的C - 2质子与重水（D₂O）中的氘进行交换，而E211Q和E168Q突变蛋白在这个部分反应中是有功能的。对于E211Q和E168Q烯醇化酶，在产物出现之前交换基本完成，这一观察结果为正常反应中的中间体提供了进一步支持。K345A烯醇化酶在磷酸乙醛酸半醛（TSP）的电离中无活性，而E168Q和E211Q蛋白都能改变互变异构状态或催化结合的TSP的电离。野生型烯醇化酶通过添加OH⁻并在C - 3处消除Cl⁻来催化（Z） - 3 - 氯 - 2 - 磷酸烯醇丙酮酸的水解。这个反应模拟了在与正常产物的逆反应中OH⁻加到磷酸烯醇丙酮酸的C - 3上的过程。所有三种突变蛋白进行这个反应的能力都有所降低。在单轮测定中，活性按K345A > E168Q >> E211Q的顺序变化。这些结果表明赖氨酸345在2 - PGA的电离中充当碱，而谷氨酸211参与反应的第二步。
