Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
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DOI：
10.1021/jacs.7b05576
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发表时间：
2017-08-02
影响因子：
15
通讯作者：
Kamerlin, Shina C. L.
中科院分区：
文献类型：
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作者：
Kulkarni, Yashraj S.;Liao, Qinghua;Kamerlin, Shina C. L.
磷酸丙糖异构酶（TIM）是一种高效的催化剂，通过E165的一般碱催化作用，可将磷酸二羟丙酮（DHAP）可逆异构化为D-甘油醛磷酸（GAP）。从历史上看，这种酶一直是理解生物催化基本原理的一个非常重要的模型系统。TIM通过能量要求很高的构象变化被激活，这有助于将两个关键疏水性残基（1170和L230）的侧链定位在E165的羧酸酯侧链上。这对于产生催化碱的疏水口袋和保持正确的活性位点结构都是至关重要的。这些残基的丙氨酸截断导致TIM的催化活性的显着下降，但实验未能提供一个完整的描述，这种钳在促进底物去质子化的作用。我们在这里进行详细的经验价键计算的TIM催化的DHAP和GAP的去质子化的野生型TIM和它的1170 A，L230 A，和1170 A/L230 A突变体，获得特殊的定量与实验一致。我们的计算提供了一个线性的自由能关系，斜率为0.8，这些TIM催化反应的活化势垒和吉布斯自由能之间。我们的结论是，这些夹紧侧链最小化底物去质子化的吉布斯自由能，反应驱动力的影响主要表现在质子转移的过渡态。我们结合以前的实验和当前的计算结果的分析，使我们能够提供一个前所未有的详细的酶催化的基态和过渡态效应的故障的概述，提供一个疏水钳磷酸丙糖异构酶的操作的分子描述。
Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (1170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild type TIM and its 1170A, L230A, and 1170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.