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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.