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

本研究的长期目标是研究重组人肝中链酰辅酶A脱氢酶(MCAD)底物特异性、构象变化和催化的分子基础。其具体目的是：(A)确定脂肪酸和辅酶-A片段在调节酶的催化效率和底物专一性中的作用；(B)建立蛋白质构象转变动力学对酶结合配体电子结构和酶催化作用的详细机制描述。中链酰辅酶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a-b脱氢。初步的光谱、动力学、热力学和建模研究导致了以下假设：(I)酰基-COAS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，(Ii)酶-配体相互作用和酶催化是通过蛋白质构象的强制性变化进行的。这些假设将通过使用UV/可见光光谱、稳态和快速动力学、等温滴定微量热法和计算机建模技术进行验证，涉及MCAD的野生型和特定位点突变。该奖项为3名研究生提供研究设施和培训，他们目前在PI的实验室工作，以获得博士/硕士学位。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。尽管已经有了几种蛋白质的三维结构，但在结合和催化过程中，负责引导和固定不同类型的配体在其同源蛋白质位置的分子作用力的作用尚不清楚。这些特性在现代生物技术中非常重要，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。来自首席研究人员实验室的初步数据表明，中链酰辅酶A脱氢酶(一种负责分解脂肪酸产生能量的酶)是一类独特的酶，表现出相当广泛的底物专一性和底物结构不同区域之间的协调。这些特征提供了一个机会，使用中链酰辅酶A脱氢酶作为一个模型系统来描述在一般酶之间的配体结合和催化过程中涉及的事件序列。

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.