Low Temperature Trapping of Intermediates in Enzyme- Catalyzed Reactions

这项研究涉及到获取酶-底物或酶-抑制剂复合体结构信息的方法开发，以及应用于慢结合过渡态类似物、蛋白酶催化的早期事件和糖苷酶催化的中间体的研究。重点研究了衰减全反射(ATR)傅里叶变换红外光谱在研究低温稳定的酶-底物中间体和酶-抑制剂复合体中的应用，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不仅有可能揭示酶-配体络合物中形成的新键，而且还有可能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毋庸置疑。分子生物学和生物技术的最新发展使人们对酶学的各个方面产生了极大的兴趣。确定酶的细节是一个主要问题。催化机理是酶-底物络合物和中间体寿命短，特别是很难获得结构信息。在极低的温度下，正常瞬变物种的寿命可以显著延长。目前正在开发一些方法，这些方法应有助于结构数据的获取。

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.