CARBONYLS IN ENZYME MECHANISM--RAMAN CHARACTERIZATION

本研究的战略目标是确定结构， 酶活性位点中底物羰基的相互作用， 将这些与酶的机制和反应性联系起来。一类 反应涉及半胱氨酸蛋白酶的酰基-酶中间体，其中 其中四种酶是与疾病有关的哺乳动物或病毒蛋白酶 状态，因此是基于知识的药物设计的目标。第二 一类反应的特点是一种对环境重要的酶，4- 氯苯甲酰辅酶A脱卤酶，参与脱氯 可能有害的氯化碳氢化合物。在所有情况下，酶- 羰基相互作用是酶机制的关键部分并且在所有情况下， 拉曼光谱提供了一种独特而强大的表征手段， 羰基的化学性质和环境。对于半胱氨酸蛋白酶， 具体目标是： * 对于哺乳动物蛋白酶，组织蛋白酶B和L（与植物蛋白酶一起 木瓜蛋白酶）和肝炎病毒蛋白酶HAV-3C，以及它们的蛋白质- 工程类似物，以在拉曼光谱中使用羰基伸缩特征， 差谱的酰基酶，以开发精确和新颖的 结构-反应性关系 * 对于钙激活的半胱氨酸蛋白酶，钙蛋白酶II，使用 结合底物的拉曼光谱，以探测钙的影响 在活跃的网站上激活。这将提供一些第一个 关于钙如何调节钙蛋白酶活性的分子信息。 * 对于半胱氨酸蛋白酶-醛过渡态类似物复合物，使用 拉曼差光谱法，以获得的振动光谱的 假定的四面体中心。这些数据的解释将提供 结构信息的过渡态类物种在点 催化转化。 所有这些研究都将得到详细的构象分析的支持 模型化合物及其同位素的振动计算 取代的类似物，以及通过底物合成和酶 动力学对于涉及4-氯苯甲酰基-CoA脱卤酶的反应， 具体目标是： * 为了使用从酶结合产物获得的拉曼数据，4- 羟基苯甲酰基-CoA和各种底物类似物，以提供一些 第一个详细的分子信息的大电子重排 发生在基底沿着反应途径和如何这些 变化是由苯甲酰羰基-活性位点相互作用介导的。

The strategic goals of this research are to define the structure and interactions of substrate carbonyl groups in enzyme active sites and to relate these to the enzyme's mechanism and reactivity. One class of reactions involves acyl-enzyme intermediates of cysteine proteases, where four of the enzymes are mammalian or viral proteases implicated in disease states and are thus targets for knowledge-based drug design. The second class of reactions features an environmentally important enzyme, 4- chlorobenzoyl-CoA dehalogenase, which participates in the dechlorination of potentially harmful chlorinated hydrocarbons. In all cases, enzyme- carbonyl interactions are a key part of enzyme mechanism and in all cases, Raman spectroscopy offers a unique and powerful means of characterizing the carbonyl's chemistry and environment. For the cysteine proteases the specific aims are: * For the mammalian proteases, cathepsins B and L (together with the plant enzyme papain) and the hepatitis viral protease HAV-3C, and their protein- engineered analogs, to use the carbonyl stretching feature in the Raman difference spectrum of acyl-enzymes to develop precise and novel structure-reactivity relationships. * For the calcium activated cysteine protease, calpain II, to use the Raman spectrum of the bound substrate to probe the effects of calcium activation on the active site. This will provide some of the first molecular information on how calcium regulates the activity of calpain. * For cysteine protease-aldehyde transition state analog complexes, to use Raman difference spectroscopy to obtain the vibrational spectrum of the putative tetrahedral center. Interpretation of these data will provide structural informational on a transition state - like species at the point of catalytic transformation. All these studies will be supported by detailed conformational analyses and vibrational calculations on model compounds and their isotopically substituted analogs, as well as by substrate synthesis and enzyme kinetics. For reactions involving 4-chlorobenzoyl-CoA dehalogenase the specific aims are: * To use the Raman data obtained from the enzyme-bound product, 4- hydroxybenzoyl-CoA, and various substrate analogs to provide some of the first detailed molecular information on the large electron rearrangements occurring in the substrate along the reaction pathway and how these changes are mediated by benzoyl carbonyl - active site interactions.