STUDIES OF ENZYME REACTION MECHANISMS

酶反应机理的研究

• 批准号: 3280688
• 负责人: STEVEN J GOULD
• 金额: $ 13.03万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-12-01 至 1987-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280688
• 关键词: RNA; S adenosylmethionine; conformation; glycine; methionine adenosyltransferase; nucleic acid metabolism; putrescine; spermidine; stereochemistry

We intend to continue our study of mechanisms of enzyme reactions where the stereochemistry is cryptic due to these reactions taking place at pro-chiral centers. Our goals are to study L-threonine dehydrogenase, aminoacetone synthase, DL-1-amino-2-propanol dehydrogenase, and DL-aminopropanol-OP phospholyase. These enzymes are all involved in the metabolism of the essential amino acid L-threonine and one of its key metabolites, aminoacetone. Significant amounts of aminoacetone are produced in the liver, and metabolic anomalies have been identified in cases of liver disease and of a number of sarcomas and carcinomas. Two of the enzymes to be studied utilize pyridoxal phosphate as coenzyme, and two use NAD. With each enzyme reaction we will prepare and use substrates chirally labeled with either deuterium or tritium, and analyze the labeling of the enzymatic products to reveal the stereochemical course of the reaction. We will also trap tritium labeled enzyme-coenzyme adducts and substrate-coenzyme adducts by in situ reduction with (3H)-sodium borohydride. These adducts will be isolated and the chirality of the labeled centers analyzed to determine whether significant conformational changes have occurred upon binding of the substrates. A recurrent and very important feature of the work proposed is the use of 3H NMR as a routine analytical tool where the very small masses of product obtainable prevent the use of deuterium labels and the more traditional 1H and 2H NMR spectroscopy. If successful, our approach will save substantial amounts of time over the currently accepted approach of extensive chemical degradations to configurational standards followed by enzymatic analyses.

我们打算继续研究酶反应的机制， 立体化学是神秘的，因为这些反应发生在 前手性中心。 我们的目标是研究L-苏氨酸脱氢酶， 氨基丙酮合酶，DL-1-氨基-2-丙醇脱氢酶，和 DL-氨基丙醇-OP磷脂酶。 这些酶都参与了 必需氨基酸L-苏氨酸的代谢及其关键之一 代谢物，氨基丙酮。 大量的氨基丙酮是 在肝脏中产生，并且代谢异常已经在 肝脏疾病和一些肉瘤和癌的病例。 待研究的两种酶利用磷酸吡哆醛作为辅酶， 两个使用NAD。 每一种酶反应我们都会准备和使用 用氘或氚手性标记的底物，并分析 对酶解产物进行标记，揭示立体化学过程 的反应。 我们还将捕获氚标记的酶-辅酶加合物 和底物-辅酶加合物，用（3 H）-钠原位还原 硼氢化物。 这些加合物将被分离，并且 分析标记的中心，以确定是否有显著的构象 在结合底物时发生变化。 拟议工作的一个经常性和非常重要的特点是使用 3 H NMR作为常规分析工具，其中非常小质量的产物 可获得的防止使用氘标记和更传统的1H 和2 H NMR光谱。 如果成功，我们的方法将节省大量的资金， 大量的时间超过目前接受的广泛的化学方法， 降解为构型标准品，然后进行酶分析。