CATALYTIC MECH:GLYOXALASE I & FORMALDEHYDE DEHYDROGENASE

催化机械：乙二醛酶 I

• 批准号: 3280212
• 负责人: Donald Creighton
• 金额: $ 16.17万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-06-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280212
• 关键词: chemical structure function; cis trans isomerization; enzyme substrate; glutathione; glyoxal; isozymes; molecular site; nuclear magnetic resonance spectroscopy; radiotracer; stereoisomer; thiols

Glyoxalase I (Glx. I) and formaldehyde dehydrogenase (FDH) are two glutathione (GSH)-dependent enzymes that operate on an equilibrium mixture of potential substrate forms composed of free aldehyde, GSH and the corresponding thiohemiacetal adduct. The overall objective of the proposed research is to determine the catalytic significance and molecular basis of the substrate specificities of these enzymes. In order to achieve this objective, the following experiments are proposed that are based, in part, on the work of the last two years. First, the nonenzymic rates of interconversion of the diasteriomers due to the physiologically important methylglyoxal-GSH thiohemiacetal will be determined by C-13 selective-inversion-recovery NMR methods. This is a follow up experiment based on the observation that the corresponding interconversion rate of the diasteriomers due to phenylglyoxal-GSH thiohemiacetal are slow (k = 10 sec-1, pH 7) in comparison to the catalytic turnover number of G1x. I. (about 500 sec-1, pH 7). This may explain the evolved capacity of the enzyme to use both diasteriomers as substrates. Second, G1x. I catalyzed interconversion of the diasteriomers due to the nonsubstrate, glyoxylic acid-GSH thiohemiacetal, will be tested for on the basis of NMR methods. A positive indication of such a catalyzed process has been obtained on the basis of a preliminary NMR line-broadening analysis of the diasteriomers in the presence of enzyme. This observation is indicative of enzyme catalyzed interconversion of the bound substrate diasteriomers before transformation to bound product. Third, the overall stereochemistry of G1x. I catalyzed interconversion of S-(D)-dithiolactoyl glutathione to the corresponding, exchange inert dithiohemiacetals will be determined in order to establish the stereochemistry of the enediol intermediate on the reaction pathway. Fourth, the ability of G1x. I to discriminate between the diasteriomers due to thiohemiacetals, formed between Alpha-ketoaldehydes and sterically hindered derivatives of GSH, will be obtained as a test of the hypothesis that positional mobility of the glutathionyl sulfur of bound substrate is required in order for the enzyme to use both diasteriomers of the normal substrate thiohemiacetals. Finally, isozymes of FDH will be tested for as a follow on preliminary observations. Contrary to previous reports, methylglyoxal is not a substrate for FDH.

Glycosidase I（Glx. I）和甲醛脱氢酶（FDH）是两种 对平衡混合物起作用的谷胱甘肽（GSH）依赖性酶 潜在的底物形式组成的游离醛，谷胱甘肽和 相应的硫代半缩醛加合物。 建议的总体目标 研究是确定催化的意义和分子基础， 这些酶的底物特异性。 为了实现这一 目的，提出以下实验，部分地， 过去两年的工作。 首先，非酶速率 由于生理学上重要的 将通过C-13测定甲基甘氨酸-GSH硫代半缩醛 选择性反转恢复NMR方法。 这是一个后续实验 根据观察， 由于苯甘氨酸-GSH硫代半缩醛的双异构体是缓慢的（k = 10 sec-1，pH 7）的催化周转数相比，G1 x。I. （约500秒-1，pH 7）。 这可能解释了进化的能力， 酶使用两种二聚体作为底物。 第二，G1 x。我催化了 由于非底物，乙醛酸， 酸-GSH硫代半缩醛，将根据NMR方法进行检测。 一 已经在实验室上获得了这种催化过程的积极迹象。 的基础上，初步NMR谱线增宽分析的双异构体， 酶的存在。 这一观察结果表明， 转化前结合底物二聚体的相互转化 绑定产品。 第三，G1 x的整体立体化学。我催化了 S-（D）-二硫代乳酰基谷胱甘肽相互转化为相应的， 将测定交换惰性二硫代半缩醛，以确定 反应途径上的环化中间体的立体化学。 第四，G1 x的能力。我必须区分由于 到硫代半缩醛，在α-酮醛之间形成， 受阻衍生物的GSH，将获得作为一个测试的假设 结合底物的谷胱甘肽硫的位置迁移率， 所需的酶，以使用两个双异构体的正常 底物硫代半缩醛。 最后，将检测FDH的同工酶， a对初步意见的后续行动。 与以前的报告相反， 丙酮醛不是FDH的底物。