Reactive Intermediates of Enzymatic Reactions

描述：(申请者描述)该提案描述了设计的研究 通过对酶催化的研究提高我们对酶催化的认识 生物重要活性中间体的形成机制 水和酶活性部位。碳酸对质子的活化 酶和小分子辅因子在酶促反应中的转移 将在两个不同的研究中进行调查。(一) α-羰基去质子化过渡态的稳定化 将探讨磷酸丙糖异构酶(TIM)对碳酸的影响，因为 大量的动力学、X射线结晶学和诱变数据未能 为这一过渡状态的稳定产生一个共识机制。上一首 对这种酶的研究在很大程度上忽视了这种利用的关键作用。 过渡态稳定时的本征底物结合能。我们 将量化去质子化的过渡态的稳定性 R-甘油醛3-磷酸(GAP)是由TIM特异性产生的 蛋白质催化剂与磷二阴离子和羰基的相互作用 衬底的一部分。接下来，使用此绑定的机制 能量将通过比较这些结合的扰动的影响来探测 诱变对GAP和a去质子酶活性的影响 缺少磷酸基团的最小底物。这些项目的目的是 相关实验是为了确定是否存在临界关闭的“手机” TIM在结合底物的磷酸盐基团上的环发生意味着允许 磷酸盐的最佳过渡态结合，或者环是否闭合 在活性中心创建质子转移的环境 本质上比在水中更有利。(2)启动 氨基酸的α-质子通过席夫碱的形成进行质子转移 与吡哆醛5‘-磷酸类似物和丙酮酰胺的加合物将是 通过测定去质子化的速率和平衡常数来量化的 这些加合物。与它们的去质子化速率常数的比较 当加合物与依赖吡哆醛的氨基酸外消旋酶结合时，将提供一种 这些质子转移的酶促速率的测量 特性不佳的碳酸。酶的认识进展 非酶反应模型研究的机制可能证明 药物设计的关键(酶抑制剂)，对新陈代谢的理解 在这方面，我们将继续致力于解决各种途径和疾病，以及解决其他与健康有关的问题。

DESCRIPTION: (Applicant's Description) The proposal describes studies designed to improve our understanding of enzymatic catalysis through investigations of the mechanism for formation of biologically important reactive intermediates in water and at enzyme active sites. The activation of carbon acids for proton transfer by enzymes and by small molecule cofactors for enzymatic reactions will be investigated in two separate studies. (1) The mechanism for stabilization of the transition state for deprotonation of alpha-carbonyl carbon acids by triosephosphate isomerase (TIM) will be probed, because the wealth of kinetic, X-ray crystallographic, and mutagenesis data has failed to produce a consensus mechanism for this transition state stabilization. Previous studies of this enzyme have largely ignored the critic role of the utilization of intrinsic substrate binding energy in transition state stabilization. We will quantify the stabilization of the transition state for deprotonation of R-gyceraldehyde 3-phosphate (GAP) by TIM that results from the specific interactions of the protein catalyst with the phosphodianion and carbonyl portions of the substrate. Next, the mechanism for utilization of this binding energy will be probed by comparing the effects of perturbation of these binding interactions by mutagenesis, on enzyme activity for deprotonation of GAP and a minimal substrate that lacks the phosphate group. The purpose of these and related experiments is to determine whether the critical closure of the "mobile loop" of TIM over the phosphate group of bound substrate occurs imply to allow optimal transition state binding of the phosphate, or whether loop closure creates an environment at the active site in which proton transfer is intrinsically more favorable than in water. (2) The activation of the alpha-protons of amino acids for proton transfer by formation of Schiff's base adducts with a pyridoxal 5'-phosphate analog and with pyruvamide will be quantified by determining rate and equilibrium constants for deprotonation of these adducts. A comparison with the rate constants for deprotonation of these adducts when bound to pyridoxal-dependent amino acid racemases will provide a measure of the enzymatic rate acceleration for proton transfer from these poorly characterized carbon acids. Advances in the understanding of enzyme mechanisms that result from model studies of nonenzymatic reactions may prove critical for drug design (enzyme inhibitors), to the understanding of metabolic pathways and diseases, and to the resolution of other health-related questions.