Reactive Intermediates of Enzymatic Reactions

酶促反应的反应中间体

• 批准号: 7422325
• 负责人: John P Richard
• 金额: $ 33.46万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7422325
• 关键词: 5-deoxypyridoxal; Acceleration; Acetaldehyde; Acids; Active Sites; Alanine; Amino Acids; Binding; Biochemical Reaction; Carbon; Carboxy-Lyases; Carboxylic Acids; Catalysis; Closure; Decarboxylation; Dependence; Deuterium; Dimerization; Disease; Distant; Drug Design; Eating; Elements; Engineering; Environment; Enzymes; Equilibrium; Evaluation; Free Energy; Generic Drugs; Glyceraldehyde 3-Phosphate; Glycine; Investigation; Isomerase; Isotopes; Lead; Measures; Metabolic Pathway; Microscopic; Modeling; Mutation; Orotidine-5' -Phosphate Decarboxylase; Phosphites; Process; Proteins; Protons; Pyridoxal Phosphate; Rate; Reaction; Relative (related person); Research Personnel; Resolution; Role; Series; Site; Solutions; Temperature; Testing; Thermodynamics; Triose-Phosphate Isomerase; Tritium; Water; analog; carbonyl group; catalyst; deprotonation; driving force; enzyme activity; enzyme mechanism; glycolaldehyde; inorganic phosphate; insight; orotidine; orotidylic acid; protein activation; protonation; research study; small molecule; transamination; vinyl ether

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.

描述（由申请人提供）：本提案描述了探索酶和吡哆醛5 '-磷酸（PLP）催化质子从碳转移的机制的实验，并合理化酶催化质子转移和脱羧反应的速率加速。磷酸丙糖异构酶（TIM）利用底物（R）-甘油醛3-磷酸的磷酸二阴离子基团的14 kcal/mol固有结合能稳定酶催化的质子转移的过渡态。我们建议TIM还利用有效的变构激活剂亚磷酸根二价阴离子的结合能来驱动构象变化，该构象变化将最小底物乙醇醛隔离在活性位点中，该活性位点具有有利于质子从碳转移的环境。我们计划：（1）检查外源性亚磷酸根二价阴离子对TIM的活化作用，使一般的简单碳酸乙醛去质子化，作为对我们假设的检验，即亚磷酸根二价阴离子与TIM的结合主要用于“设计”具有有利于烯醇化的环境的活性位点。(2)探讨TIM关键“移动的环”闭合在简单碳酸质子转移中的作用。(3)探索乳清酸核苷5 '-磷酸（OMP）的磷酸二价阴离子基团与OMP-脱羧酶相互作用诱导的构象变化是否以类似的方式用于“设计”有利于底物脱羧过渡态稳定的活性位点。我们还提出了一个简单的吡哆醛5 '-磷酸类似物的甘氨酸和丙氨酸的α-氨基质子的活化的特点，并评估隧道的质子通过非酶质子转移在碳的反应障碍的重要性。了解这些过程中的水是必不可少的，以评估其在酶催化作用。从酶和非酶反应的机械研究中了解酶催化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。