Low Temperature Trapping of Intermediates in Enzyme- Catalyzed Reactions

酶催化反应中中间体的低温捕获

• 批准号: 9107070
• 负责人: Anthony Fink
• 金额: $ 23.8万
• 依托单位: University of California-Santa Cruz
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9107070&HistoricalAwards=false
• 关键词: Low; Temperature; Trapping; Intermediates; Enzyme

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.

建议的研究包括结合方法开发获取酶-底物或酶-抑制剂复合体的结构信息，以及对B-内酰胺酶进行定点突变研究，以确定活性中心残基在催化和抑制中的作用。傅里叶变换红外光谱(FTIR)，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(如过渡态类似物)复合体。FTIR不仅有可能揭示酶-配体复合体中形成的新键，而且还可能揭示酶的键变形和极化以及构象变化。这些方法将被应用于几个系统，以增加我们对酶催化和抑制的分子基础的理解。病原菌对β-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，耐药的原因是感染剂产生的B型内酰胺酶的存在，这种酶能水解B型内酰胺，使其不能作为抗生素使用。本研究旨在加深我们对B-内酰胺酶催化机理的了解。本研究将利用定点突变技术来确定几个被认为在催化中起重要作用的残基的作用。最终目标是设计基于机制的B-内酰胺酶抑制剂，它也将起到抗生素的作用。