Substrate Recognition and Activation in Beta-Oxidation

点击翻译按钮获取中文摘要

DESCRIPTION (Applicant's abstract): This proposal will determine the structure of conjugated ligands bound to enoyl-CoA hydratase and acyl-CoA dehydrogenase, two enzymes in the fatty acid n-oxidation pathway. Two questions will be addressed: (1) Can the molecular basis for the alterations in substrate electronic structure required for efficient catalysis be determined and (2) can the enzyme-substrate interactions responsible for preferential binding of one substrate conformer over another be identified? These questions stem from two proposals, namely: (1) that changes in the electronic structure of conjugated substrate/product analogs that occur upon binding to the enzymes reflect enzyme-substrate interactions in place to stabilize charge rearrangement as the reaction proceeds and (2) that the stereospecificity of the enzyme catalyzed reaction results from the preferential reaction of one bound conformer rather than the binding of a single substrate conformer to the enzyme. The experimental approaches involve modulating the size of the ligand or active site and modulating specific enzyme-ligand interactions using enzyme mutagenesis or ligand synthesis. The impact of these alterations on the distribution of bound ligand conformers, the stereochemistry of the reaction and changes in structure-reactivity will be assessed using vibrational and NMR spectroscopy in combination with enzyme kinetics. The identification and quantitation of enzyme-substrate interactions that correctly orient the substrate and stabilize charge redistribution will provide a basis for inhibitor design. Inhibitors of beta-oxidation enzymes have potential application as novel therapeutics for treating pathological conditions such as reperfusion-injury of the ischemic heart following a myocardial infarction and myocardial dysfunction in diabetes.