CATALYTIC MECH:GLYOXALASE I & FORMALDEHYDE DEHYDROGENASE

催化机械：乙二醛酶 I

• 批准号: 3280216
• 负责人: Donald Creighton
• 金额: $ 13.53万
• 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/3280216
• 关键词: chemical structure function; cis trans isomerization; enzyme substrate; glutathione; glyoxal; isozymes; molecular site; nuclear magnetic resonance spectroscopy; radiotracer; stereoisomer; thiols; tritium

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.

乙二醛酶 I (Glx. I) 和甲醛脱氢酶 (FDH) 是两种 在平衡混合物上运行的谷胱甘肽 (GSH) 依赖性酶 由游离醛、GSH 和 相应的半硫缩醛加合物。 拟议的总体目标 研究的目的是确定催化意义和分子基础 这些酶的底物特异性。 为了实现这一目标 目的，提出以下实验，部分基于： 关于近两年的工作。 首先，非酶速率 由于重要的生理学原因，非对映异构体发生相互转化 甲基乙二醛-GSH硫半缩醛将由C-13测定 选择性反转恢复核磁共振方法。 这是后续实验 根据观察，相应的相互转化率 由苯基乙二醛-GSH 硫半缩醛引起的非对映异构体缓慢（k = 10 sec-1，pH 7）与 G1x 的催化周转数相比。我。 （约 500 秒-1，pH 7）。 这可以解释进化能力 酶使用两种非对映异构体作为底物。 第二，G1x。我催化了 由于非底物乙醛酸导致非对映体相互转化 酸-GSH硫半缩醛，将基于NMR方法进行测试。 一个 已经获得了这种催化过程的积极迹象 对非对映异构体进行初步 NMR 谱线展宽分析的基础 酶的存在。 该观察结果表明酶催化 转化前结合底物非对映异构体的相互转化 绑定产品。 第三，G1x的整体立体化学。我催化了 S-(D)-二硫代乳酰谷胱甘肽相互转化为相应的， 将测定交换惰性二硫半缩醛，以便建立 反应途径中烯二醇中间体的立体化学。 第四，G1x的能力。我要区分非对映异构体 硫代半缩醛，在α-酮醛和空间之间形成 GSH 的受阻衍生物，将作为假设检验获得 结合底物的谷胱甘肽硫的位置迁移率为 为了使酶能够使用正常的两种非对映异构体，需要 底物硫代半缩醛。 最后，FDH 的同工酶将被测试为 初步观察的后续。 与之前的报道相反， 甲基乙二醛不是 FDH 的底物。