A Disease Causing Mutation in Acyl-CoA Dehydogenase

这项拟议研究的长期目标是阐明致病突变对人肝中链酰辅酶A脱氢酶(MCAD)蛋白质稳定性和催化性质的影响。在短期内，我们建议调查受Lys304-&Gt；Glu(K304E)突变影响的MCAD的某些特性，Lys304-&Gt；Glu(K304E)突变是最显著的(致病)脂肪酸代谢的先天错误之一。本研究的具体目的是：(A)确定该酶对K304E突变的动力学和热力学稳定性。(B)确定催化受损的分子基础，以及该酶对K304E突变的底物专一性的变化。MCAD中的Lys-304-&Gt；Glu(K304E)突变在人类患者中表现为Reye综合征样症状。我们在野生型和K304E突变的MCAD催化的反应的结构-功能方面的初步比较数据导致了以下假设：(I)K304E突变由于在亚基间区引入净负电荷而使酶蛋白不稳定。(Ii)K304E突变会影响酶的催化效率和底物专一性，这是由于酶环境中静电场的改变所致。这些假设将通过使用UV/可见光、荧光和CD光谱、稳态和快速动力学、等温滴定微量热技术来验证，涉及野生型和K304E突变酶。

The long term goal of the proposed research is to elucidate the influence of disease-causing mutations on protein stability and catalytic properties of human liver medium chain acyl-CoA dehydrogenase (MCAD). In short term, we propose to investigate selected properties of MCAD, which are affected by the Lys304->Glu (K304E) mutation, one of the most prominent (disease causing) inborn errors of fatty acid metabolism. The specific aims of the proposed research is to accomplish the following: (a) Determine the kinetic and thermodynamic stability of the enzyme upon K304E mutation. (b) Ascertain the molecular basis of impaired catalysis, and changes in the substrate specificity of the enzyme upon K304E mutation. The Lys-304->Glu (K304E) mutation in MCAD is manifested in Reye syndrome like symptoms in human patients. Our preliminary comparative data on the structural-functional aspects of the wild-type and K304E mutant MCAD-catalyzed reactions have led to the following hypotheses: (i) the K304E mutation destabilizes the enzyme protein due to incorporation of a net negative charge at the inter-subunit region. (ii) the K304E mutation impairs the catalytic efficiency and substrate specificity of the enzyme due to alteration in the electrostatic field of the enzyme site environment. These hypotheses will be tested via the use of uv/visible, fluorescence, and CD spectroscopy, steady-state and rapid kinetics, isothermal titration microcalorimetric techniques, involving wild-type and K304E mutant enzymes.