Activation of Enzymes for Catalysis: The Role of Substrate-Induced Structural Changes

催化酶的激活：底物诱导的结构变化的作用

基本信息

批准号：
9198549
负责人：
John P Richard
金额：
$ 33.35万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9198549
关键词：
Acceleration Active Sites Affinity Architecture Binding Biochemical Reaction Buffaloes Carboxy-Lyases Catalysis Chemicals Complex Data Decarboxylation Dependence Deuterium Diphosphates Disease Drug Design Elements Enzyme Activation Enzymes Glycerol-3-Phosphate Dehydrogenase Glycols Goals Health Hydrogen Bonding Isomerase Isotopes Kinetics Ligands Mechanics Metabolic Diseases Metabolic Pathway Modeling Movement Mutagenesis Mutation NADH Nature Organism Phosphites Proteins Protocols documentation Publishing Pyrimidine Reaction Resolution Role Side Site Solvents Specificity Structural Protein Temperature Testing Therapeutic Triose-Phosphate Isomerase Water analog carbanion catalyst design enzyme mechanism enzyme model experimental study glycolaldehyde grasp hydroxyl group inhibitor/antagonist innovation inorganic phosphate interest isopentenyl pyrophosphate orotidylic acid public health relevance quantum reaction rate small molecule tetrahydrofuran

项目摘要

DESCRIPTION (provided by applicant): Enzymes are distinguished from small molecule catalysts by their highly evolved reaction mechanisms that enable the utilization of binding interactions with non-reacting portions of the substrate for transition state stabilization. Innovative protocols developed at Buffalo will be used to test the hypothesis that specificity in transition state binding is obtained by utilization of the intrinsic binding energy of substrate fragments - such as a phosphodianion, pyrophosphotrianion, or ribofuranosyl ring - to drive energetically demanding and structurally complex changes from an inactive open enzyme to a catalytically active caged Michaelis complex with substrate. The relationship between the extraordinary 1017-fold rate acceleration for decarboxylation of orotidine 5'-monophosphate (OMP) catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) and the extensive movements of a phosphodianion gripper loop and a pyrimidine umbrella that accompany formation of the Michaelis complex will be examined. (1) The effects of multiple mutations at both the gripper loop and the pyrimidine umbrella will be examined, to determine whether enzyme activation is the result of cooperative closure of these two protein structural elements. (2) Activation of OMPDC toward catalysis of decarboxylation of 5-fluoroorotate, the ultimate truncated substrate, by exogenous cis-tetrahydrofuran-3,4-diol and phosphite dianion will be examined. (3) The activating nature of the interactions between OMPDC and the ribofuranosyl hydroxyl groups of OMP will be probed in mutagenesis experiments and in studies of substrate analogs lacking these hydroxyl groups. The effect of site-directed mutations on dianion activation of the reduction of the truncated substrate glycolaldehyde by NADH catalyzed by glycerol 3-phosphate dehydrogenase (GPDH) will be determined. The data will be compared with those from published studies of OMPDC and triosephosphate isomerase (TIM), in order to define the essential features of the active site architectures of TIM, OMPDC and GPDH that enable dianion activation of reactions proceeding through chemically diverse transition states. The temperature dependence of the primary deuterium kinetic isotope effect on the phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde by NADH/NADD will be examined. It will be determined whether these isotope effects are consistent with a classical model for hydride transfer or with a more complex model involving quantum mechanical tunneling through the barrier. The kinetic parameters for isomerization of isopentenyl monophosphate and for incorporation of deuterium from solvent D2O into the truncated neutral substrate 2-methylpropene catalyzed by isopentenyl diphosphate isomerase (IDI) will be determined. Activation of IDI-catalyzed deuterium exchange into the truncated substrate by the isohypophosphate trianion substrate piece will be examined, in order to test the proposal that binding interactions between IDI and the substrate pyrophosphotrianion group are utilized to stabilize the transition state for formation of an enzyme- bound tertiary carbocation.

描述（由申请人提供）：酶与小分子催化剂的区别在于其高度进化的反应机制，能够利用与底物非反应部分的结合相互作用来实现过渡态稳定，将用于测试布法罗开发的创新方案。假设过渡态结合的特异性是通过利用底物片段（例如磷酸二价阴离子、焦磷酸三价阴离子或呋喃核糖基环）的内在结合能来获得的从无活性的开放酶到具有底物的催化活性笼式米氏复合物的能量要求高且结构复杂的变化由乳清苷 5'-单磷酸脱羧酶催化的乳清苷 5'-单磷酸 (OMP) 脱羧速率非凡的 1017 倍加速之间的关系。（OMPDC）以及伴随广泛形成的磷酸二价阴离子夹环和嘧啶伞的运动(1) 将检查夹环和嘧啶伞的多个突变的影响，以确定酶激活是否是这两个蛋白质结构元件协同闭合的结果。 OMPDC 通过外源顺式四氢呋喃-3,4-二醇和亚磷酸二价阴离子催化最终截短的底物 5-氟乳清酸脱羧(3) 将在诱变实验和缺乏这些羟基的底物类似物的研究中探讨 OMPDC 和 OMP 呋喃核糖基羟基之间相互作用的激活性质。定点突变对双阴离子激活的影响。将测定3-磷酸甘油脱氢酶(GPDH)催化的NADH对截短底物乙醇醛的还原，并比较数据。与已发表的 OMPDC 和磷酸三糖异构酶 (TIM) 研究中的结果相结合，以确定 TIM、OMPDC 和 GPDH 活性位点结构的基本特征，这些特征能够通过化学不同的过渡态进行双阴离子激活反应。将检查初级氘动力学同位素对亚磷酸双阴离子激活的 GPDH 催化的 NADH/NADD 还原乙醇醛的影响。确定这些同位素效应是否与氢化物转移的经典模型或涉及通过势垒的量子力学隧道效应的更复杂的模型一致。异戊烯基二磷酸异构酶 (IDI) 催化的 2-甲基丙烯将被测定为 IDI 催化的氘交换的活化。将检查由异次磷酸三阴离子底物片截断的底物，以测试利用 IDI 和底物焦磷酸三阴离子基团之间的结合相互作用来稳定形成酶结合的叔碳正离子的过渡态的提议。