METALLOENZYME DYNAMICS AND MECHANISM

酶与其底物的动态相互作用沿 定义明确的催化途径，由多个基本的 台阶。当这一途径被穿越时，瞬时酶-底物(E。s) 中间产物形成了。为了充分了解其致病机制 酶的催化作用，至关重要的是这两种酶的结构 以及这些中间体中的底物被阐明。在过去, 对E.事实证明，S的中介人很难，因为他们的短暂 寿命长，浓度低。此外，一般的方法能够 在溶液中提供有关结构的信息还没有开发出来。 拟议的研究结合了当前研究的两个领域，以克服 这些问题。零度以下的温度和低温溶剂将用于 在高浓度下积累中间体一段时间 允许他们进行光谱研究。然后光谱学将被用于 研究底物或酶具有发色性的中间体 以产生部分活性中心的振动光谱。这项研究 将专注于金属酶，因为它们通常有发色中心 非常适合于共振拉曼研究。在一系列 通过实验，得到了水解物的RR谱。 亮氨酸氨肽酶催化的发色肽和二硫酯底物 将进行研究以检测底物在 催化作用。在其他实验中，中间体的RR谱在 发色酶对无色底物的氧化作用 将检测萝卜过氧化物酶，以检测结构的变化 参与催化作用的血红素基团。

The dynamic interaction of an enzyme with its substrate occurs along a well-defined catalytic pathway that consists of multiple, elementary steps. As this pathway is traversed, transient enzyme-substrate (E . S) intermediates are formed. In order to fully understand the mechanism of enzyme catalysis, it is essential that the structures of both the enzyme and substrate in each of these intermediates be elucidated. In the past, studies of E . S intermediates have proven difficult because of their brief lifetime andlow concentration. Furthermore, general methods capable of providing information about structure in solution have not been developed. The proposed research combines two areas of current research to overcome these problems. Subzero temperatures and cryosolvents will be used to accumulate intermediates in high concentrations for periods of time that permit their spectroscopic study. The spectroscopy will then be used to study intermediates in which either the substrate or enzyme is chromophoric to yield vibrational spectra of parts of the active site. This research will focus on metalloenzymes because they often have chromophoric centers that are ideally suited for resonance Raman studies. In one series of experiments, the rR spectra of intermediates in the hydrolysis of chromophoric peptide and dithioester substrates by leucine amino-peptidase will be studied to detect structural changes in the substrate during catalysis. In other experiments, rR spectra of intermediates formed during the oxidation of colorless substrates by the chromophoric enzyme horse radish peroxidase will be examined to detect changes in the structure of the heme group as it participates in catalysis.