Enzyme architecture: modeling the operation of a hydrophobic clamp in catalysis by triosephosphate isomerase

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 (I170 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 ...磷酸丙糖异构酶（Triosephosphate isomerase，TIM）是一类高效催化剂，可通过第165位谷氨酸残基（E165）的广义碱催化（general base catalysis）机制，催化磷酸二羟丙酮（dihydroxyacetone phosphate，DHAP）与d-甘油醛磷酸（d-glyceraldehyde phosphate，GAP）之间的可逆异构反应。长期以来，该酶一直是探究生物催化基本原理的关键模型体系。TIM需通过耗能的构象变化完成激活，该过程可将两个关键疏水残基——异亮氨酸170（I170）与亮氨酸230（L230）的侧链，精准定位至E165的羧酸盐侧链上方。这一定位对于构建催化碱基所需的疏水口袋、维持活性位点的正确架构均至关重要。将这两个残基突变为丙氨酸会显著降低TIM的催化活性，但现有实验尚未能完整阐明该钳位结构促进底物去质子化的具体作用机制。本文开展了针对TIM催化DHAP与GAP去质子化过程的详细经验价键计算（empirical valence bond calculations）……