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CATALYTIC MECH:GLYOXALASE I & FORMALDEHYDE DEHYDROGENASE

催化机械：乙二醛酶 I

基本信息

批准号：
3280217
负责人：
Donald Creighton
金额：
$ 11.59万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE COUNTY
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-06-01 至 1990-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3280217
关键词：
chemical structure function cis trans isomerization enzyme mechanism enzyme substrate glutathione glyoxal isozymes molecular site nuclear magnetic resonance spectroscopy radiotracer stereoisomer thiols tritium

项目摘要

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.

乙二酸酶I(GLX.I)和甲醛脱氢酶(FDH)是两种在平衡混合物上工作的谷胱甘肽(GSH)依赖的酶可能的底物形式包括游离醛、GSH和相应的硫代半缩醛加合物。建议的总体目标是研究是为了确定其催化意义和分子基础这些酶的底物特性。为了实现这一目标目的，提出了以下实验，这些实验部分基于，关于过去两年的工作。首先，非酶的速率生理学上重要的非对映异构体的相互转化用C-13测定甲基乙二醛-谷胱甘肽半缩醛选择反转恢复核磁共振方法。这是一个后续实验根据观察到的相应的相互转换率苯乙醛-谷胱甘肽半缩醛的非对映异构体反应缓慢(k=10 SEC-1，pH 7)与G1X的催化转化率进行比较。我。 (约500秒-1，pH值7)。这可能解释了人类进化的能力使用两种非对映异构体作为底物的酶。第二，G1X。我催化了非底物乙醛引起的非对映异构体的相互转化酸-谷胱甘肽半缩醛，将在核磁共振方法的基础上进行测试。一个这种催化过程的积极迹象已经在非对映异构体的初步核磁共振谱线展宽分析酶的存在。这一观察结果表明酶被催化。结合底物非对映异构体在转化前的相互转化来装订产品。第三，G1X的整体立体化学。我催化了 S-(D)-二硫代乳酸基谷胱甘肽相互转化为相应的，交换惰性二硫代半缩醛将被测定以建立反应路径上的烯二醇中间体的立体化学。第四，G1X的能力。我要区别对待非对映异构体到硫代半缩醛，在α-酮醛和立体之间形成 GSH的受阻衍生物，将作为对该假说的检验结合底物的谷胱甘肽基硫的位置迁移率为酶使用正常的两个非对映异构体所必需的底物硫代半缩醛。最后，将检测外佣的同工酶是否为AS 后续的初步观察。与之前的报道相反，甲基乙二醛不是FDH的底物。