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（由适用提供）：酶通过其高度进化的反应机制与小分子催化剂区分开来，从而使结合相互作用与底物的非反应部分的结合相互作用进行过渡态稳定。在布法罗开发的创新协议将用于检验以下假设：过渡状态结合的特异性是通过利用底物碎片的固有结合能（例如磷酸二苯二醇，吡咯磷酸二苯二醇，或核腺呋喃糖基环）的，从而驱动型号的型号迫切型牛肉症，从而驱动了型号的catteration cattration cattration catty，c磷酸磷酸化或核呋喃糖基环会驱动型号的型号。甲磷酸甲苯二羧酸甲苯二羧酸甲基化（OT磷酸盐（OMP））的非凡1017倍加速度之间的关系是由5'-单磷酸甲磷酸甲苯二甲酸甲酰脱羧酶（OMPDC）催化的确定酶激活是否是这两个蛋白质结构元素合作关闭的结果。 （2）通过外源性顺式四氢呋喃-3,4-二醇和磷酸二醇和磷酸二醇和磷酸二醇和磷酸二醇和磷酸二醇和磷酸二醇和磷酸二醇和磷酸二醇和磷酸二醇和磷矿dianion的最终截短底物的5-氟甲二羧酸脱羧的激活。 （3）在诱变实验和缺乏这些羟基的底物类似物的研究中，OMPDC与OMP核呋喃糖基羟基之间相互作用的激活性质将进行探测。将确定位置定向突变对通过3-磷酸甘油脱氢酶（GPDH）催化的NADH截短的底物甘油醛还原的dianion激活的影响。将将数据与已发表的OMPDC和三卫磷酸异构酶（TIM）的已发表研究的数据进行比较，以定义TIM，OMPDC和GPDH的主动位点体系结构的基本特征，以使反应通过化学多样性过渡状态进行反应的dianion激活。将检查NADH/NADD的主要动力学同位素效应对磷酸二苯二体激活的GPDH催化降低的温度依赖性。将确定这些同位素效应是否与氢化物转移的经典模型是一致的，还是涉及通过屏障的量子机械隧穿的更复杂的模型。异戊烯基单磷酸异构化的动力学参数和氘企业从溶剂d2O到截短的中性中性底物2-甲基二苯甲酸甲基甲基甲基甲基甲基甲基甲基甲基二磷酸异构酶（IDI）的催化。将检查IDI催化氘化的氘交换为截短的底物，由等磷酸磷酸盐三角底物进行检查，以测试IDI与底物焦距磷酸磷脂组之间的结合相互作用的提议可用于稳定Enzyme-Bondiary Carboction tertersy terterter terterterterter的过渡状态。