Substrate Recognition and Activation in Beta-Oxidation

描述（申请人摘要）：本提案将确定结构 与烯酰辅酶A水合酶和酰基辅酶A脱氢酶结合的共轭配体， 脂肪酸N-氧化途径中的两种酶。两个问题将是 解决：（1）可以改变底物的分子基础 确定有效催化所需的电子结构，以及（2）可以 酶-底物相互作用负责优先结合一个 底物构象异构体超过另一个被确定？ 这些问题源于两个建议，即：（1）改变 共轭底物/产物类似物的电子结构， 与酶的结合反映了酶-底物的相互作用， 随着反应的进行，稳定电荷重排，以及（2） 酶催化反应的立体专一性是由 一种结合构象异构体的优先反应，而不是一种结合构象异构体的优先反应。 单底物构象的酶。 实验方法涉及调节配体或活性配体的大小。 使用酶定位和调节特异性酶-配体相互作用 诱变或配体合成。这些变化对 结合配体构象的分布，反应的立体化学 和结构反应性的变化将使用振动和NMR进行评估 光谱学与酶动力学相结合。 酶-底物相互作用的鉴定和定量， 正确地定向衬底并稳定电荷再分布将提供 抑制剂设计的基础。β-氧化酶的抑制剂具有 作为治疗病理性疾病的新疗法的潜在应用 例如缺血性心脏的再灌注损伤的病症， 糖尿病心肌梗死和心肌功能障碍。

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.