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Dynamic and Catalysis by Alcohol Dehydrogenases

醇脱氢酶的动力学和催化

基本信息

批准号：
7677831
负责人：
BRYCE V PLAPP
金额：
$ 26.85万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7677831
关键词：
Active Sites Affect Affinity Alcohol dehydrogenase Alcoholism Alcohols Amino Acid Substitution Amino Acids Benzaldehyde Benzyl Alcohols Binding Binding Sites Carbon Catalysis Catalytic Domain Chemicals Coenzymes Complex Conserved Sequence Coupled Data Development Dissociation Distant Drug Design Elements Enzymes Equilibrium Equus caballus Ethanol Metabolism Evolution Global Change Hydrogen Kinetics Ligand Binding Ligands Liquid substance Liver Medical Molecular Conformation Motion Multienzyme Complexes Muscle Rigidity Mutate Mutation NADH Parents Pathology Pathway interactions Phase Principal Investigator Probability Proteins Protons Reaction Relative (related person)Research Research Personnel Resolution Role Rotation Scaffolding Protein Series Side Site Site-Directed Mutagenesis Specificity Structure System Techniques Temperature Tertiary Protein Structure Testing Therapeutic Agents Translations Variant X-Ray Crystallography Zinc analog base carbonyl compound catalyst chemical binding computer studies design dimer enzyme structure flexibility fluidity improved inhibitor/antagonist interest oxidation programs protein structure reaction rate scaffold three dimensional structure vibration

项目摘要

DESCRIPTION (provided by applicant): The long-term objectives of this research are to determine the roles of protein structure, flexibility, adaptability and dynamics in enzyme catalysis. The overall hypothesis is that residues at or distant from the active site contribute to catalysis by affecting protein motions and fluidity, conformational changes, and subunit interactions. Horse liver alcohol dehydrogenase is a good system for studying structural and dynamics effects on catalysis. Three-dimensional structures of various complexes have been determined by X-ray crystallography for two conformational states, and rate and dissociation constants for each step in the mechanism, including the chemical step (hydride transfer), can be estimated from steady-state and transient kinetics. Site-directed mutagenesis will be used to substitute amino acid residues in different regions of the enzyme, and the effects on the catalytic mechanism, structure and dynamics will be quantitatively evaluated. (1) Residues in the hinge region between the catalytic and coenzyme binding domains and the domain contact regions will be altered in order to change the rate and extent of the conformational change, and allosteric interactions through the subunits will be studied with heterodimeric enzymes. (2) Amino acid residues that may contribute to protein promoting vibrations and residues buried in core regions will be mutated in order to change the protein fluidity and dynamics. "Extraneous" structural elements or Q-loops will be deleted to test the role of the protein scaffold. (3) Three-dimensional structures of wild-type and mutated enzymes complexed with substrate analogs will be determined by X-ray crystallography at high resolution, and dynamic information will be extracted by analysis of translation, libration, screw-rotation displacements (TLS parameters) for domains and subdomains that cooperate in catalysis. The dynamics hypothesis will be supported if the directions and amplitudes of motion are correlated with rates of hydride transfer. The kinetic, structural and dynamic results should improve our understanding of catalysis and facilitate rational drug design. Redesign of enzymes for medical and industrial applications should become more "rational" when the connections between protein structure and catalysis are better understood. Development of new catalysts based on protein scaffolds will benefit from a better understanding of protein motions and dynamics. Furthermore, the design of therapeutic agents must incorporate information about protein motions, as specificities are affected by amino acid residues that are distant from the active site. Studies on substrate and inhibitor specificities of alcohol dehydrogenases suggest that active sites are adaptable and that binding affinities are not easily explained with a static structure. Design of better inhibitors for treating the pathology of alcoholism depends on understanding the dynamics of catalysis by the rate-limiting enzyme in the pathway of alcohol metabolism.

描述（由申请人提供）：本研究的长期目标是确定蛋白质结构、灵活性、适应性和动态在酶催化中的作用。总体假设是，位于或远离活性位点的残基通过影响蛋白质运动和流动性、构象变化和亚基相互作用来促进催化。马肝乙醇脱氢酶是研究催化结构和动力学效应的良好系统。各种配合物的三维结构已通过 X 射线晶体学确定了两种构象状态，并且可以根据稳态和瞬态动力学估计该机制中每个步骤（包括化学步骤（氢化物转移））的速率和解离常数。将利用定点诱变来替换酶不同区域的氨基酸残基，并定量评估其对催化机制、结构和动力学的影响。 (1)催化结构域和辅酶结合结构域之间的铰链区以及结构域接触区中的残基将被改变，以改变构象变化的速率和程度，并且将用异二聚酶研究通过亚基的变构相互作用。 (2)可能有助于蛋白质促进振动的氨基酸残基和埋藏在核心区域的残基将被突变，以改变蛋白质的流动性和动力学。 “无关的”结构元件或 Q 环将被删除，以测试蛋白质支架的作用。（3）通过高分辨率X射线晶体学确定与底物类似物复合的野生型和突变酶的三维结构，并通过分析催化配合的域和子域的平移、振动、螺杆旋转位移（TLS参数）来提取动态信息。如果运动的方向和幅度与氢化物转移速率相关，则动力学假设将得到支持。动力学、结构和动力学结果应该提高我们对催化的理解并促进合理的药物设计。当更好地理解蛋白质结构和催化之间的联系时，用于医学和工业应用的酶的重新设计应该变得更加“合理”。基于蛋白质支架的新催化剂的开发将受益于对蛋白质运动和动力学的更好理解。此外，治疗剂的设计必须纳入有关蛋白质运动的信息，因为特异性受到远离活性位点的氨基酸残基的影响。对醇脱氢酶的底物和抑制剂特异性的研究表明，活性位点具有适应性，并且结合亲和力不容易用静态结构来解释。设计更好的治疗酒精中毒病理的抑制剂取决于了解酒精代谢途径中限速酶的催化动力学。