Reactive Intermediates of Enzymatic Reactions

描述（由申请人提供）：本提案描述了探索酶和5'-磷酸吡哆醛（PLP）催化质子从碳转移的机理，并合理化酶催化质子转移和脱羧反应的速率加速的实验。三磷酸异构体酶（TIM）利用底物(R)- 3-磷酸甘油醛的磷酸二氢基14千卡/摩尔的固有结合能来稳定酶催化质子转移的过渡态。我们建议TIM还利用强效变构活化剂亚磷酸酯离子的结合能来驱动构象变化，将最小的底物乙醇醛固定在一个有利于碳质子转移的活性位点。我们计划：(1)研究外源性亚磷酸腺苷对TIM的激活作用，以促进一般简单碳酸乙醛的去质子化，作为我们假设的检验，即亚磷酸腺苷与TIM的结合主要是为了“工程”一个具有有利于烯醇化环境的活性位点。(2)探讨了TIM临界“移动环”的关闭在简单碳酸质子转移中的作用。(3)探究orotidine 5'-phosphate （OMP）的磷酸基与OMP-脱羧酶相互作用引起的构象变化是否以类似的方式被用于“工程”一个有利于底物脱羧过渡态稳定的活性位点。我们还建议用一个简单的吡哆醛5'-磷酸类似物来表征甘氨酸和丙氨酸的α -氨基质子的活化，并评估质子穿过反应势垒对碳上非酶质子转移的重要性。了解水中的这些过程对于评估它们在酶催化中的作用至关重要。从对酶和非酶反应的机理研究中获得的对酶催化的理解的进展可能对药物设计、对代谢途径和疾病的理解以及对其他健康相关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.