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: 8810144
• 负责人: Anthony Fink
• 金额: $ 17.88万
• 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: 1988
• 资助国家: 美国
• 起止时间: 1988-07-15 至 1991-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8810144&HistoricalAwards=false
• 关键词: Low; Temperature; Trapping; Intermediates; Enzyme

This research involves a combination of method development for the acquisition of structural information concerning enzyme- substrate or enzyme-inhibitor complexes, and application to the study of slow-binding transition-state analog inhibitors, early events in protease catalysis and intermediates in glycosidase catalysis. The focus of the investigation is on the application of attenuated total reflectance (ATR) Fourier transfer infrared spectroscopy to study enzyme-substrate intermediates, stabilized at low temperatures, and enzyme- inhibitor complexes, and the use of immobilized enzymes and stopped-flow kinetics in cryoenzymology studies. FT-IR has the potential to reveal not only new bonds formed in enzyme-ligand complexes, but also bond deformations and polarization. The importance of enzymes in numerous areas of science, medicine and industry needs no elaboration. Recent developments in molecular biology and biotechnology have led to a greatly increased interest in all aspects of enzymology. A major problem in determining the details of an enzyme. Catalytic mechanism is the short lifetime of enzyme - substrate complexes and intermediates, making it particularly difficult to obtain structural information. The lifetimes of normal transient species can be significantly increased at sintably low temperatures. The development of some methods which should facilitate the acquisition of structural data are underway here.

本研究包括获取酶-底物或酶-抑制剂复合物结构信息的方法开发，以及应用于慢结合过渡态类似物抑制剂、蛋白酶催化早期事件和糖苷酶催化中间体的研究。研究的重点是应用衰减全反射（ATR）傅立叶转移红外光谱来研究酶-底物中间体，在低温下稳定，酶-抑制剂复合物，以及固定化酶和停止流动动力学在低温酶学研究中的应用。FT-IR不仅可以揭示酶-配体复合物中形成的新键，还可以揭示键的变形和极化。酶在科学、医学和工业的许多领域中的重要性无需赘述。分子生物学和生物技术的最新发展极大地增加了人们对酶学各个方面的兴趣。确定酶的细节的一个主要问题。催化机理是酶-底物配合物和中间体的寿命很短，使得获得结构信息特别困难。正常瞬态物种的寿命可以在一定的低温下显著增加。一些有助于获取结构数据的方法正在研究中。