STUDIES OF ENZYME REACTION MECHANISMS

我们打算继续研究酶反应的机制，其中 立体化学是神秘的，因为这些反应发生在 亲手性中心。我们的目标是研究L苏氨酸脱氢酶， 氨基丙酮合成酶、DL-1-氨基-2-丙醇脱氢酶和 DL-氨基丙醇-OP磷酸水解酶。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 在肝脏中产生，代谢异常已在 肝脏疾病以及一些肉瘤和癌症的病例。 被研究的两种酶利用磷酸吡哆醛作为辅酶， 两个人使用NAD。对于每一种酶反应，我们都会准备和使用 用氘或氚手性标记的衬底，并分析 酶产物的标记揭示立体化学过程 反应的结果。我们还将捕获氚标记的酶-辅酶加合物 和底物-辅酶加合物的(~3H)-钠原位还原 硼氢化物。这些加合物将被分离出来，并且手性 分析标记中心以确定显著构象 底物结合时发生了变化。 拟议工作的一个反复出现的和非常重要的特点是使用 ~3H核磁共振作为常规分析工具，其中非常小质量的产品 可获得的防止使用氚的标签和更传统的1H 和~2H核磁共振波谱。如果成功，我们的方法将节省大量资金 超过当前接受的广泛化学方法的时间量 降解到构型标准，然后进行酶分析。

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.