CARBONYLS IN ENZYME MECHANISM--RAMAN CHARACTERIZATION

本研究的战略目标是确定结构和 酶活性部位底物羰基与TO的相互作用 这些都与酶的机制和反应有关。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。 国家，因此是以知识为基础的药物设计的目标。第二 一类反应的特点是一种对环境重要的酶，4- 参与脱氯的氯苯甲酰辅酶A脱卤酶 潜在有害的氯代烃。在所有情况下，酶- 羰基相互作用是酶机制的关键部分，在所有情况下， 拉曼光谱提供了一种独特而强大的手段来表征 羰基的化学和环境。对于半胱氨酸蛋白酶来说 具体目标是： *哺乳动物的蛋白水解酶、组织蛋白酶B和L(连同植物 木瓜酶)和肝炎病毒蛋白酶HAV-3C，以及它们的蛋白- 经过改造的类似物，在拉曼光谱中使用羰基伸展特性 酰基酶的差示光谱技术发展精密新奇 结构-反应性关系。 *对于钙激活的半胱氨酸蛋白酶，Calain II，使用 结合底物的拉曼光谱研究钙离子的作用 在活动站点上激活。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，请使用 利用拉曼差分光谱技术获得分子的振动光谱 假定为四面体中心。对这些数据的解释将提供 关于过渡态类物种在这一点上的结构信息 催化转化的结果。 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 取代类似物，以及底物合成和酶 运动学。对于涉及4-氯苯甲酰辅酶A脱卤酶的反应 具体目标是： *使用从酶结合产物获得的拉曼数据，4- 羟基苯甲酰辅酶A和各种底物类似物提供了一些 关于大电子重排的第一个详细的分子信息 沿着反应路径在底物中发生，以及这些 改变是由苯甲酰基-活性中心相互作用引起的。

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.