A Disease Causing Mutation in Acyl-CoA Dehydogenase

该研究的长期目标是阐明致病突变对人肝脏中链酰基辅酶A脱氢酶（MCAD）蛋白质稳定性和催化特性的影响。在短期内，我们建议研究MCAD的选定特性，这些特性受到Lys 304->Glu（K304 E）突变的影响，Lys 304->Glu（K304 E）突变是脂肪酸代谢的最突出（致病）先天性缺陷之一。所提出的研究的具体目标是完成以下内容：（a）确定K304 E突变后酶的动力学和热力学稳定性。(b)确定受损催化的分子基础，以及K304 E突变后酶底物特异性的变化。MCAD中的Lys-304->Glu（K304 E）突变表现为人类患者中的Reye综合征样症状。我们对野生型和K304 E突变体MCAD催化反应的结构-功能方面的初步比较数据得出以下假设：（i）K304 E突变使酶蛋白不稳定，这是由于在亚基间区域掺入净负电荷。(ii)由于酶位点环境的静电场发生改变，K304 E突变会损害酶的催化效率和底物特异性。这些假设将通过使用紫外/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，涉及野生型和K304 E突变酶进行测试。

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.