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

• 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
• 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

 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稳定过渡态。布法罗开发的创新方案将用于测试这样的假设：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环）的内在结合能来获得的，以驱动从无活性的开放酶到具有催化活性的笼状米氏复合物与底物的能量要求高且结构复杂的变化。将检查由乳清苷 5'-单磷酸脱羧酶 (OMPDC) 催化的乳清苷 5'-单磷酸 (OMP) 脱羧速率非凡的 1017 倍加速与伴随米氏复合体形成的磷酸二价阴离子夹环和嘧啶伞的广泛运动之间的关系。 (1) 将检查夹环和嘧啶伞上的多个突变的影响，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。 (2)将检查外源顺式四氢呋喃-3,4-二醇和亚磷酸二价阴离子对OMPDC的激活作用，以催化最终截短的底物5-氟乳清酸脱羧。 (3) OMPDC 和 OMP 呋喃核糖基羟基之间相互作用的激活性质将在诱变实验和缺乏这些羟基的底物类似物的研究中进行探讨。将确定定点突变对甘油3-磷酸脱氢酶(GPDH)催化的NADH还原截短的底物乙醇醛的双阴离子激活的影响。这些数据将与已发表的 OMPDC 和磷酸三糖异构酶 (TIM) 研究中的数据进行比较，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上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效应对亚磷酸二价阴离子激活的 GPDH 催化的 NADH/NADD 乙醇醛还原的温度依赖性。将确定这些同位素效应是否与氢化物转移的经典模型一致，或者是否与涉及穿过势垒的量子力学隧道效应的更复杂的模型一致。将确定异戊烯基单磷酸异构化和由异戊烯基二磷酸异构酶 (IDI) 催化的氘从溶剂 D2O 掺入截短的中性底物 2-甲基丙烯的动力学参数。将检查异次磷酸三阴离子底物片对 IDI 催化的氘交换至截短底物的激活，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酶结合的三级碳正离子形成的过渡态的提议。