ENZYME MECHANISMS IN THREE CARBON METABOLISM

三碳代谢中的酶机制

• 批准号: 3287111
• 负责人: John W Kozarich
• 金额: $ 12.14万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 1989-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287111
• 关键词: acyltransferase; affinity labeling; enzyme substrate; formates; glutathione; glyoxal; halogenation; hydrogen transport; isomerase; lactates; nuclear magnetic resonance spectroscopy

This grant seeks continued support for our research on the mechanism of enzyme action. Specifically, we propose to investigate three enzymes which are involved in the metabolism of three-carbon units. Our work on glyoxalase I will focus on the identification of the active site base responsible for proton transfer using some of the principles of our recent finding of "mirror-image" catalysis by glyoxalase I. In addition, other studies are designed to probe the nature of the partitioning reaction of halomethylglyoxals by the enzyme. We will attempt to determine the contributions of product release from the enzyme and halogen orientation on the partitioning reaction. Our studies on lactate racemse will focus on two major issues. First, we will investigate the question of proton versus hydride transfer in the racemization step. We will accomplish this using many of the techniques that we have established in the glyoxalase I studies. Second, we will address the question of substrate activation in this system. There has been some speculation that lactate racemase may involve a covalent enzyme-substrate complex or a "charged" enzyme. Extreme difficulty in the purification and stabilization of this enzyme has prevented the resolution of this issue. We propose to develop a general method for the detection of a "charged" enzyme which we call anhydride detection by intermolecular oxygen scrambling. The technique relies on the implied radomization of carboxylate oxygens in an acyl exchange mechanism. Our technique will involve a novel utilization of 18O perturbations on 13C NMR resonances. This method will be extended to other enzymes where "charged" forms and anhydride intermediates have been proposed. Finally, we would like to initiate studies on elucidating the molecular mechanism of pyruvate-formate lyase, an enzyme which bears some similarity to the two discussed above in terms of ambiguity of electron flow. We propose studies designed to determine the direction of bond cleavage, the intermediates formed on the enzyme, and the function of enzyme activation by SAM, flavodoxin, and Fe(II)-protein complex.

这项拨款旨在继续支持我们对 酶的作用 具体来说，我们建议研究三种酶， 参与了三碳单位的新陈代谢。 我们的工作 glycoproteinase I将重点放在活性位点碱基的鉴定上 负责质子转移使用我们最近的一些原则， 发现glycoprotein酶I的“镜像”催化作用。 此外，其他 研究的目的是探讨分配反应的性质， 卤甲基乙二醛的酶。 我们将尝试确定 从酶和卤素方向释放产物的贡献 分配反应。 我们对乳酸消旋体的研究主要集中在两个方面。 一是 将调查质子与氢化物转移的问题， 外消旋化步骤。 我们将使用许多技术来实现这一点 我们在glycoprotein I研究中已经建立的。 二是 解决了该系统中底物活化的问题。 一直 有人推测乳酸消旋酶可能涉及共价键， 酶-底物复合物或“带电”酶。 极端困难， 这种酶的纯化和稳定化阻止了 这个问题。 我们建议开发一种通用的检测方法， 一种“带电”酶，我们称之为分子间酸酐检测 氧气干扰 该技术依赖于隐含的随机化 羧酸氧在酰基交换机制。 我们的技术将 涉及在13 C NMR共振上18 O扰动新利用。 这种方法将被扩展到其他酶，其中“带电”形式， 已经提出了酸酐中间体。 最后，我们希望启动研究，阐明分子 具有某些相似性的酶--琥珀酸-甲酸裂解酶的作用机制 在电子流的不确定性方面与上面讨论的两个不同。 我们 提出旨在确定键断裂方向的研究， 酶上形成的中间体，以及酶的活化功能 通过SAM、黄素氧还蛋白和Fe（II）-蛋白复合物。