A Disease Causing Mutation in Acyl-CoA Dehydogenase

一种导致酰基辅酶A脱氢酶突变的疾病

• 批准号: 6316139
• 负责人: D. K SRIVASTAVA
• 金额: $ 10.58万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6316139
• 关键词: acyl coA dehydrogenases; chemical kinetics; circular magnetic dichroism; colorimetry; enzyme substrate; fatty acid metabolism; fluorescence spectrometry; gene mutation; glutamates; inborn lipid /lipoprotein disorder; intermolecular interaction; ionic bond; liver; lysine; microcalorimetry; molecular pathology; molecular site; protein folding; protein structure function; structural biology; thermodynamics; thermostability; ultraviolet spectrometry

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.

本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）蛋白稳定性和催化性能的影响。短期内，我们建议研究MCAD的一些特性，这些特性受Lys304->Glu （K304E）突变的影响，这是最突出的（引起疾病的）脂肪酸代谢先天性错误之一。本研究的具体目的是完成以下工作：(a)确定K304E突变后酶的动力学和热力学稳定性。(b)确定催化功能受损的分子基础，以及K304E突变后酶的底物特异性的变化。MCAD中的Lys-304->Glu （K304E）突变在人类患者中表现为Reye综合征样症状。我们对野生型和K304E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) K304E突变由于在亚基间区域引入净负电荷而使酶蛋白不稳定。（ii） K304E突变由于酶位点环境静电场的改变而削弱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进行测试，涉及野生型和K304E突变型酶。

项目成果

Two-prong inhibitors for human carbonic anhydrase II.

人碳酸酐酶 II 的双管齐下抑制剂。

• DOI: 10.1021/ja047271k
• 发表时间: 2004
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Roy,BidhanC;Banerjee,AbirL;Swanson,Michael;Jia,XiaoG;Haldar,ManasK;Mallik,Sanku;Srivastava,DK
• 通讯作者: Srivastava,DK