PDF(Pubmed)
Abstract：
The P168 and I172 side chains sit at the heart of the active site of triosephosphate isomerase (TIM) and play important roles in the catalysis of the isomerization reaction. The phosphodianion of substrate glyceraldehyde 3-phosphate (GAP) drives a conformational change at the TIM that creates a steric interaction with the P168 side chain that is relieved by the movement of P168 that carries the basic E167 side chain into a clamp that consists of the hydrophobic I172 and L232 side chains. The P168A/I172A substitution at TIM from Trypanosoma brucei brucei (TbbTIM) causes a large 120,000-fold decrease in kcat for isomerization of GAP that eliminates most of the difference in the reactivity of TIM compared to the small amine base quinuclidinone for deprotonation of catalyst-bound GAP. The I172A substitution causes a > 2-unit decrease in the pKa of the E167 carboxylic acid in a complex to the intermediate analog PGA, but the P168A substitution at the I172A variant has no further effect on this pKa. The P168A/I172A substitutions cause a 5-fold decrease in Km for the isomerization of GAP from a 0.9 kcal/mol stabilization of the substrate Michaelis complexes. The results show that the P168 and I172 side chains play a dual role in destabilizing the ground-state Michaelis complex to GAP and in promoting stabilization of the transition state for substrate isomerization. This is consistent with an important role for these side chains in an induced fit reaction mechanism [Richard, J. P. (2022) Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution. Biochemistry 61, 1533-1542].
摘要：
P168和I172侧链位于磷酸丙糖异构酶（TIM）活性位点的中心，在异构化反应的催化中起重要作用。底物甘油醛3-磷酸（GAP）的磷酸二阴离子驱动TIM处的构象变化，该构象变化与P168侧链产生空间相互作用，该相互作用通过携带基本E167侧链的P168移动进入由疏水I172和L232侧链组成的钳夹而得以缓解。布氏锥虫（TbbTIM）在TIM上的P168A/I172A取代导致GAP异构化的kcat大幅下降120,000倍，这消除了TIM与小胺碱奎宁酮相比的反应性差异的大部分用于去质子化催化剂结合的GAP。I172A取代导致中间体类似物PGA的复合物中E167羧酸的pKa降低>2-单元，但是I172A变体处的P168A取代对该pKa没有进一步的影响。P168A/I172A取代导致GAP异构化的Km从底物Michaelis复合物的0.9kcal/mol稳定化降低了5倍。结果表明，P168和I172侧链在使基态米氏络合物与GAP失稳和促进底物异构化的过渡态稳定方面起着双重作用。这与这些侧链在诱导配合反应机制中的重要作用一致[Richard，J.P.(2022)配体驱动的构象变化在酶进化中的作用。生物化学61,1533-1542]。
