Department of Chemistry , University at Buffalo, SUNY , Buffalo , New York 14260-3000 United States.
Department of Chemistry , University of Illinois at Urbana-Champaign , 600 S Mathews Avenue , Urbana , Illinois 61801 , United States.
J Am Chem Soc. 2018 Jul 5;140(26):8277-8286. doi: 10.1021/jacs.8b04367. Epub 2018 Jun 21.
We report pH rate profiles for k and K for the isomerization reaction of glyceraldehyde 3-phosphate catalyzed by wildtype triosephosphate isomerase (TIM) from three organisms and by ten mutants of TIM; and, for K for inhibition of this reaction by phosphoglycolate trianion (I). The pH profiles for K show that the binding of I to TIM (E) to form EH·I is accompanied by uptake of a proton by the carboxylate side-chain of E165, whose function is to abstract a proton from substrate. The complexes for several mutants exist mainly as E·I at high pH, in which cases the pH profiles define the p K for deprotonation of EH·I. The linear free energy correlation, with slope of 0.73 ( r = 0.96), between k/ K for TIM-catalyzed isomerization and the disassociation constant of PGA trianion for TIM shows that EH·I and the transition state are stabilized by similar interactions with the protein catalyst. Values of p K = 10-10.5 were estimated for deprotonation of EH·I for wildtype TIM. This p K decreases to as low as 6.3 for the severely crippled Y208F mutant. There is a correlation between the effect of several mutations on k/ K and on p K for EH·I. The results support a model where the strong basicity of E165 at the complex to the enediolate reaction intermediate is promoted by side-chains from Y208 and S211, which serve to clamp loop 6 over the substrate; I170, which assists in the creation of a hydrophobic environment for E165; and P166, which functions in driving the carboxylate side-chain of E165 toward enzyme-bound substrate.
我们报告了三种生物体来源的野生型磷酸丙糖异构酶（TIM）和 TIM 的十个突变体催化甘油醛-3-磷酸异构化反应的 k 和 K 的 pH 值速率曲线，以及磷酸烯醇丙酮酸三阴离子（I）抑制该反应的 K。K 的 pH 值曲线表明，I 与 TIM（E）结合形成 EH·I 伴随着 E165 侧链羧基吸收质子，其功能是从底物中提取质子。对于几个突变体，复合物主要以 E·I 的形式存在于高 pH 值下，在这种情况下，pH 值曲线定义了 EH·I 的去质子化 p K。TIM 催化的异构化的 k/ K 与 PGA 三阴离子与 TIM 解离常数之间的线性自由能相关性，斜率为 0.73（r = 0.96），表明 EH·I 和过渡态通过与蛋白质催化剂的相似相互作用稳定。野生型 TIM 中 EH·I 的去质子化 p K 值估计为 10-10.5。对于严重受损的 Y208F 突变体，p K 值降至低至 6.3。几个突变对 k/ K 和 EH·I 的 p K 值的影响之间存在相关性。结果支持了一种模型，其中 E165 在与烯醇化物反应中间体的复合物中的强碱性由 Y208 和 S211 的侧链促进，它们用于将 loop 6 夹在底物上；I170 有助于为 E165 创造疏水环境；P166 则在驱动 E165 的羧基侧链朝向酶结合底物方面发挥作用。
