Molecular Basis of Substrate Specificity, Conformational Changes, and Catalytic Efficiency in Medium Chain Acyl-CoA Dehydrogenase

中链酰基辅酶 A 脱氢酶的底物特异性、构象变化和催化效率的分子基础

• 批准号: 9904416
• 负责人: D. Srivastava
• 金额: $ 33万
• 依托单位: North Dakota State University Fargo
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904416&HistoricalAwards=false
• 关键词: Molecular; Basis; Substrate; Specificity; Conformational

9904416SrivastavaThe long term goal of this research is to investigate the molecular basis of substrate specificity, conformational changes, and catalysis in recombinant human liver medium chain acyl-CoA dehydrogenase (MCAD). The specific aims are: (A) To determine the roles of fatty acid and coenzyme-A fragments of acyl-CoAs in modulating the catalytic efficiency and substrate specificity of the enzyme, and (B) To establish a detailed mechanistic description of the roles of dynamics of the protein conformational transitions on the electronic structures of the enzyme-bound ligands, and the enzyme catalysis. Medium chain acyl-CoA dehydrogenase (MCAD) is a flavoenzyme, which catalyzes the a-b dehydrogenation of acyl-CoA substrates during the mitochondrial b-oxidative pathway. Preliminary spectroscopic, kinetic, thermodynamic, and model building studies have led to the following hypotheses: (i) The fatty acid and coenzyme-A fragments of acyl-CoAs coordinate in stabilizing the ground and transition state structures during the enzyme catalysis, and (ii) The enzyme-ligand interactions and the enzyme catalysis proceed via obligatory changes in the protein conformation. These hypotheses will be tested via the use of uv/visible spectroscopy, steady-state and rapid kinetics, isothermal titration microcalorimetry, and computergraphic model building techniques, involving the wild-type and selected site-specific mutations of MCAD. This award provides research facilities and training to 3 graduate students, who are currently working in the PI's laboratory toward obtaining their Ph.D./M.S. degrees. This research will unravel several intrinsic features of the role of the protein structures in modulating functions. Despite the availability of the 3-dimensional structures of several proteins, the role of the molecular forces responsible for steering and immobilizing different types of ligands at their cognate protein sites during the course of binding and catalysis is not clearly understood. Such properties are of utmost importance in modern biotechnology, particularly from the point of view of designing novel enzymes, pesticides, and other commercially valuable products. Preliminary data from the principal investigator's laboratory have led to the suggestion that medium chain acyl-CoA dehydrogenase (an enzyme responsible for the breakdown of fatty acids to generate energy) is one of a class of unique enzymes, which exhibit a fairly broad range of substrate specificity and coordinate among different regions of the substrate structures. These features provide an opportunity to use medium chain acyl-CoA dehydrogenase as a model system for delineating the sequence of events involved during the course of ligand binding and catalysis among enzymes in general.

本研究的长期目标是研究重组人肝中链酰基辅酶A脱氢酶（MCAD）的底物特异性、构象变化和催化作用的分子基础。具体目标是：（A）确定酰基辅酶A的脂肪酸和辅酶A片段在调节酶的催化效率和底物特异性中的作用;（B）建立蛋白质构象转变动力学对酶结合配体的电子结构和酶催化作用的详细机理描述。中链酰基辅酶A脱氢酶（MCAD）是一种黄素酶，在线粒体b-氧化途径中催化酰基辅酶A底物的a-b脱氢。初步的光谱学、动力学、热力学和模型构建研究已经导致了以下假设：（i）酰基辅酶A的脂肪酸和辅酶A片段在酶催化过程中协调稳定基态和过渡态结构，以及（ii）酶-配体相互作用和酶催化通过蛋白质构象的强制性变化进行。这些假设将通过使用紫外/可见光谱，稳态和快速动力学，等温滴定微量热法，和计算机图形模型构建技术，涉及野生型和选定的位点特异性突变的MCAD进行测试。 该奖项为3名研究生提供研究设施和培训，他们目前正在PI的实验室获得博士学位。M.S.度这项研究将揭示蛋白质结构在调节功能中的作用的几个内在特征。尽管几种蛋白质的三维结构的可用性，在结合和催化过程中，负责操纵和固定不同类型的配体在其同源蛋白质位点的分子力的作用还不清楚。这些性质在现代生物技术中至关重要，特别是从设计新型酶、杀虫剂和其他有商业价值的产品的角度来看。 来自主要研究者实验室的初步数据表明，中链酰基辅酶A脱氢酶（一种负责分解脂肪酸以产生能量的酶）是一类独特的酶之一，其表现出相当广泛的底物特异性，并在底物结构的不同区域之间进行协调。这些功能提供了一个机会，使用中链酰基辅酶A脱氢酶作为一个模型系统，用于描绘过程中的配体结合和酶之间的催化作用的过程中所涉及的事件的顺序一般。