METALLOENZYME DYNAMICS AND MECHANISM

酶与其底物的动态相互作用发生在沿着 明确的催化途径，包括多个，基本的 步 当这条途径通过时，瞬时酶底物（E。S）的 中间体形成。 为了全面了解 酶催化，这是必要的，这两种酶的结构 以及这些中间体中的每一种的底物。 在过去， 研究E . S中间体已被证明是困难的，因为它们的简短 寿命长、浓度低。 此外，能够 提供关于溶液中结构的信息还没有被开发出来。 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 在一段时间内积累高浓度的中间体， 允许他们进行光谱研究。 光谱学将用于 研究底物或酶是发色团的中间体 以产生活性位点的部分的振动光谱。 本研究 我将集中研究金属酶，因为它们通常具有发色中心 非常适合共振拉曼研究。 在一系列的 实验中，在水解的中间体的rR光谱 亮氨酸氨基肽酶的发色肽和二硫酯底物 将进行研究，以检测在基板的结构变化， 催化作用 在其他实验中，对在反应过程中形成的中间体的rR谱进行了测量。 发色酶马对无色底物的氧化 萝卜过氧化物酶将被检查，以检测结构的变化 血红素基团，因为它参与催化。

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.