Dynamic and Catalysis by Alcohol Dehydrogenases

醇脱氢酶的动力学和催化

• 批准号: 7483781
• 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
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483781
• 关键词: Active Sites; Affect; Affinity; Alcohol dehydrogenase; Alcoholism; Alcohols; Amino Acid Substitution; Amino Acids; Benzaldehyde; Benzyl Alcohol; 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; Pliability; Principal Investigator; Probability; Proteins; Protons; Range; Rate; 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; fluidity; improved; inhibitor/antagonist; interest; oxidation; programs; protein structure; reaction rate; scaffold; size; three dimensional structure; vibration

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、适应性和动力学在酶催化中的作用。总的假设是，活性位点附近或附近的残基通过影响蛋白质的运动和流动性、构象变化和亚基相互作用来促进催化作用。马肝醇脱氢酶是研究马肝醇脱氢酶的结构和动力学效应的良好体系。用x射线晶体学测定了两种构象状态下各种配合物的三维结构，并从稳态和瞬态动力学估计了机制中包括化学步骤（氢化物转移）在内的每个步骤的速率和解离常数。将利用位点导向诱变取代酶不同区域的氨基酸残基，并定量评价对催化机制、结构和动力学的影响。(1)催化和辅酶结合结构域与结构域接触区域之间的铰链区域的残基将被改变，从而改变构象变化的速率和程度，并且通过亚基与异二聚体酶的变构相互作用将被研究。(2)可能促进蛋白质振动的氨基酸残基和埋在核心区域的残基会发生突变，从而改变蛋白质的流动性和动力学。“外来的”结构元件或q环将被删除，以测试蛋白质支架的作用。(3)利用x射线晶体学高分辨率确定野生型和突变型酶与底物类似物络合的三维结构，并通过分析协同催化的结构域和子结构域的平移、振动、螺旋旋转位移（TLS参数）提取动态信息。如果运动方向和振幅与氢化物转移速率相关，则动力学假设将得到支持。动力学、结构和动力学方面的研究结果有助于提高我们对催化的认识，促进药物的合理设计。当对蛋白质结构和催化作用之间的联系有了更好的了解时，用于医疗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型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，治疗剂的设计必须包含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。对醇脱氢酶底物和抑制剂特异性的研究表明，活性位点是可适应的，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对酒精代谢途径中限速酶的催化动力学的理解。