Low Temperature Trapping of Intermediates in Enzyme- Catalyzed Reactions

本研究涉及获取有关酶底物或酶抑制剂复合物的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、蛋白酶催化的早期事件和糖苷酶催化的中间体的研究的组合。 研究重点是应用衰减全反射 (ATR) 傅里叶转移红外光谱来研究低温稳定的酶-底物中间体和酶-抑制剂复合物，以及固定化酶和停流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，还可以揭示键变形和极化。 酶在科学、医学和工业众多领域的重要性无需赘述。 分子生物学和生物技术的最新发展导致人们对酶学各个方面的兴趣大大增加。 确定酶细节的主要问题。 催化机制是酶-底物复合物和中间体的寿命短，使得获得结构信息特别困难。 在极低的温度下，正常瞬态物质的寿命可以显着增加。 这里正在开发一些有助于获取结构数据的方法。

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.