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.
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