Reactive Intermediates of Enzymatic Reactions

描述(申请人提供)：本提案描述了探索酶和5‘-磷酸吡哆醛(PLP)催化碳质子转移的机理，并使酶催化的质子转移和脱羧化反应的速度加快合理化的实验。磷酸丙糖异构酶(TIM)利用底物(R)-3-磷酸甘油醛的磷酸二阴离子基团14kcal/mol的固有结合能稳定过渡态，进行酶催化的质子转移。我们认为，TIM还利用了强有力的变构活化剂亚磷酸二阴离子的结合能来驱动构象变化，将最低限度的底物乙醇醛隔离在一个有利于质子从碳转移的活性中心。我们计划：(1)检测外源性亚磷酸二阴离子对普通简单碳酸乙醛去质子化的激活作用，以检验我们的假设，即亚磷酸二阴离子与TIM的结合主要是为了在有利于烯醇化的环境中“设计”一个活性部位。(2)探讨TIM关键“移动环”的闭合在简单碳酸质子转移中的作用。(3)探讨5‘-磷酸鸟苷(OMP)的磷酸二阴离子基团与OMP-脱羧酶相互作用所引起的构象变化是否被用来“设计”有利于底物脱羧基过渡态稳定的活性部位。我们还建议表征一个简单的吡哆醛5‘-磷酸类似物对甘氨酸和丙氨酸的α-氨基质子的激活，并评估质子通过反应屏障对碳上非酶质子转移的重要性。对水中这些过程的了解对于评估它们在酶催化中的作用是至关重要的。从酶和非酶反应的机理研究中了解酶催化的进展可能被证明对药物设计、新陈代谢途径和疾病的理解以及其他与健康有关的问题的解决至关重要。

DESCRIPTION (provided by applicant): 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.