Low Temperature Trapping of Intermediates in Enzyme- Catalyzed Reactions

拟议的研究包括获取酶-底物或酶-抑制剂复合物结构信息的方法开发，以及对b -内酰胺酶的定点诱变研究，以确定活性位点残基在催化和抑制中的作用。傅里叶转移红外（FTIR）光谱，特别是衰减全反射（ATR） FTIR，将用于研究酶-底物中间体（在低温下稳定）和酶-抑制剂（例如过渡态类似物）配合物。FTIR不仅有潜力揭示酶-配体复合物中形成的新键，而且有潜力揭示键的变形和极化，以及酶的构象变化。这些方法将应用于几个系统，以增加我们对酶催化和抑制的分子基础的理解。病原菌对b -内酰胺类抗生素的耐药性是一个重大的国际卫生问题。在大多数情况下，耐药的原因是感染原产生的b -内酰胺酶的存在，它水解b -内酰胺，使其作为抗生素无效。本研究旨在增加我们对b -内酰胺酶催化机制的认识。位点定向诱变将用于确定几种残基的作用，这些残基已被提出在催化中很重要。最终目标是设计基于机制的b -内酰胺酶抑制剂，它也将作为抗生素起作用。

The proposed research involves a combination of method development for the acquisition of structural information about enzyme-substrate or enzyme-inhibitor complexes, and site-directed mutagenesis studies on B-lactamase to determine the role of active-site residues in catalysis and inhibition. Fourier Transfer Infrared (FTIR) spectroscopy, especially attenuated total reflectance (ATR) FTIR, will be used to study enzyme-substrate intermediates (stabilized at low temperatures) and enzyme-inhibitor (e.g. transition state analogs) complexes. FTIR has the potential to reveal not only new bonds formed in enzyme-ligand complexes, but also bond deformations and polarization, as well as conformational changes in the enzyme. These methods will be applied to several systems to increase our understanding of the molecular basis for enzyme catalysis and inhibition . Resistance of pathogenic organisms toward B-lactam antibiotics is a major international health problem. In the majority of cases the reason for the resistance is the presence of B-lactamases produced by the infectious agent, which hydrolyze the B-lactam rendering it ineffective as a antibiotic . This research is aimed at increasing our understanding of the mechanism of catalysis of B-lactamases.m Site-directed mutagenesis will be used to determine the role of several residues which have been proposed to be important in catalysis. The ultimate goal is the design of mechanism- based inhibitors of the B-lactamases which will also function as antibiotics.