Reactive Intermediates of Enzymatic Reactions

该方案描述了探索碳原子质子转移催化机理的实验。 酶和吡哆醛5‘-磷酸(PLP)，并使酶催化的速度加快合理化 质子转移和脱羧基反应。磷酸丙糖异构酶(TIM)利用14千卡/摩尔 底物(R)-3-磷酸甘油醛的磷二阴离子基团的内禀结合能 酶催化质子转移过渡态的稳定化。我们建议TIM还利用 有效的变构激活剂亚磷酸二阴离子的结合能驱动构象变化 将最低限度的底物乙醇醛隔离在活性部位，其环境有利于 碳的质子转移。我们计划：(1)检测外源性亚磷酸根双阴离子对TIM的激活作用 对于普通的简单碳酸乙醛的去质子化，作为对我们假设的测试， 亚磷酸二阴离子与TIM的结合主要用于在具有 倾向于同盟化。(2)探讨了TIM关键“移动环”的闭合在质子转移中的作用。 简单碳酸。(3)探讨分子间相互作用引起的构象变化 5‘-磷酸鸟苷(OMP)的磷酸二阴离子与OMP-脱羧酶一起用于类似的 以“设计”活性中心的方式，有利于底物脱羧基的过渡态稳定。我们 还建议用一种简单的方法来表征甘氨酸和丙氨酸的α-氨基质子的活化 5‘-磷酸吡哆醛类似物，并评估质子通过反应隧穿的重要性 碳上非酶质子转移的障碍。了解水中的这些过程是至关重要的。 以评估它们在酶催化中的作用。酶催化作用的研究进展 这种对酶和非酶反应的机理研究可能被证明对药物设计至关重要， 了解代谢途径和疾病，并解决其他与健康有关的问题。

This proposal describes experiments to probe the mechanism of catalysis of proton transfer from carbon by enzymes and by pyridoxal 5'-phosphate (PLP), and to rationalize the rate acceleration for enzyme-catalyzed proton transfer and decarboxylation reactions. Triosephosphate isomerase (TIM) utilizes the 14 kcal/mol intrinsic binding energy of the phosphodianion group of the substrate (R)-glyceraldehyde 3-phosphate in stabilization of the transition state for enzyme-catalyzed proton transfer. We propose that TIM also utilizes the binding energy of the potent allosteric activator phosphite dianion to drive a conformational change that sequesters the minimal substrate glycolaldehyde in an active site with an environment that is favorable for proton transfer from carbon. We plan to: (1) Examine the activation of TIM by exogenous phosphite dianion toward deprotonation of the generic simple carbon acid acetaldehyde, as a test of our hypothesis that the binding of phosphite dianion to TIM serves primarily to "engineer" an active site with an environment that favors enolization. (2) Probe the role of closure of the critical "mobile loop" of TIM in proton transfer from simple carbon acids. (3) Probe whether the conformational change induced by interactions of the phosphodianion group of orotidine 5'-phosphate (OMP) with OMP-decarboxylase are utilized in a similar manner to "engineer" an active site that favors transition state stabilization for substrate decarboxylation. We also propose to characterize the activation of the alpha-amino protons of glycine and alanine by a simple pyridoxal 5'-phosphate analog, and to evaluate the importance of tunneling of the proton through the reaction barrier for nonenzymatic proton transfer at carbon. An understanding of these processes in water is essential to an evaluation of their role in enzymatic catalysis. Advances in the understanding of enzyme catalysis from such mechanistic studies on enzymes and nonenzymatic reactions may prove critical for drug design, to the understanding of metabolic pathways and diseases, and to the resolution of other health-related questions.