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

 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，该反应机制使得能够利用与底物的非反应部分的结合相互作用来实现过渡态稳定。在布法罗开发的创新方案将被用来测试的假设，即特异性过渡态结合是通过利用底物片段的固有结合能-如磷酸二阴离子，焦磷酸三阴离子，或呋喃核糖环-驱动能量需求和结构复杂的变化，从一个无活性的开放酶的催化活性笼米氏复合物与基板。乳清酸核苷5 '-单磷酸脱羧酶（OMPDC）催化的乳清酸核苷5'-单磷酸（OMP）脱羧反应的1017倍速率加速与伴随米氏复合物形成的磷酸二阴离子夹环和嘧啶伞的广泛运动之间的关系将被检查。(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合作闭合的结果。(2)将检查OMPDC对5-氟乳清酸（最终截短的底物）通过外源性顺式-四氢呋喃-3，4-二醇和亚磷酸酯二价阴离子的脱羧催化的活化。(3)OMPDC和OMP的呋喃核糖基羟基之间的相互作用的活化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还原截短底物乙醇醛的二价阴离子活化的影响。这些数据将与已发表的OMPDC和磷酸丙糖异构酶（TIM）的研究进行比较，以定义TIM，OMPDC和GPDH的活性位点结构的基本特征，使二价阴离子活化的反应通过化学上不同的过渡态进行。研究了亚磷酸根二阴离子活化的GPDH催化的乙醇醛还原反应中初级氘动力学同位素效应的温度依赖性。将确定这些同位素效应是否与氢化物转移的经典模型或涉及通过势垒的量子力学隧穿的更复杂的模型一致。将测定异戊烯基单磷酸异构化和异戊烯基二磷酸异构酶（IDI）催化的氘从溶剂D2 O掺入截短的中性底物2-甲基丙烯的动力学参数。将检查IDI催化的氘交换通过异连二磷酸三阴离子底物片进入截短的底物的活化，以测试IDI和底物焦磷酸三阴离子基团之间的结合相互作用用于稳定过渡态以形成酶结合的叔碳阳离子的提议。

 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.