Substrate Recognition and Activation in Beta-Oxidation

描述(申请者摘要)：本建议书将确定结构 与烯丙基辅酶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。将有两个问题 解决：(1)底物变化的分子基础 有效催化所需的电子结构被确定，以及(2)可以 酶与底物的相互作用导致1-甲基-4-甲氧基异丁基酮的优先结合 底物构象比另一种构象要确定吗？ 这些问题源于两个建议，即：(1) 共轭底物/产物类似物的电子结构 与酶的结合反映了酶与底物的相互作用 随着反应的进行，稳定的电荷重排和(2) 酶催化反应的立体特异性源于 一个结合的构象的优先反应而不是一个结合的 酶的单底物构象。 实验方法包括调节配体或活性的大小。 利用酶来定位和调节特定的酶-配体相互作用 诱变或配体合成。这些变化对世界经济的影响 结合配体构象的分布、反应的立体化学 结构-反应性的变化将使用振动和核磁共振进行评估 光谱学与酶动力学相结合。 酶-底物相互作用的鉴定和定量 正确地定向衬底和稳定的电荷重新分布将提供 为缓蚀剂的设计奠定了基础。β-氧化酶的抑制剂有 作为治疗病理疾病的新疗法的潜在应用 心脏缺血后的再灌注损伤等情况 糖尿病合并心肌梗死和心功能不全。

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.