MECHANISMS OF ENZYME ACTION

酶的作用机制

• 批准号: 3287114
• 负责人: John W Kozarich
• 金额: $ 17.96万
• 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 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287114
• 关键词: anaerobic bacteria; catalyst; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; enzyme substrate; formates; glycolysis; glyoxal; lyase; prokaryote; pyruvates

This competitive renewal application seeks continued support for our major research effort directed toward the elucidation of fundamental new chemistry of enzyme and inhibitor action. During the next funding period, we will continue our work in three major areas: studies on the activation and chemical mechanism of E. coli pyruvate formate-lyase, further investigations on the mechanism of halide eliminations through intervening aromatic rings by enzymes which catalyze carbanionic mechanisms and some final studies on the stereospecificity of glyoxalase I catalysis. Our effort will focus on the key enzyme of prokaryotic anaerobic glycolysis, pyruvate formate-lyase (PFL). Recent work, including our own, has provided suggestive evidence that the reaction may proceed by an enzymatically unprecedented homolytic cleavage of pyruvate. Our approach will involve a thorough study of the mode inactivation of PFL by hypophosphite, acetylphosphinate and propargylic acid. The latter two were recently discovered by us to be extremely potent inactivators of the enzyme. The novel mode of activation of PFL will also be investigated. These studies will require a broad effort with expertise in synthesis, enzyme purification, stable and radio isotope analyses, high field NMR and EPR studies. We have recently demonstrated that novel quinodimethane intermediates are generated from p-halomethyl-substituted aromatic substrates by enzymes which proceed by putative carbanionic mechanisms. The phenomenon has been established for glyoxalase I, benzoyl-formate decarboxylase and mandelate racemase. These studies will now be extended to the flavin-dependent D- amino acid oxidase and, in an attempt to determine if radical mechanisms also can catalyze these eliminations, to monoamine oxidase. This work is of particular significance to the area of drug design and enzyme inactivation by quinone methide intermediates. Finally, we will execute some final experiments to definitely establish that glyoxalase I has the unique and evolutionarily significant ability to catalyze a non-stereo-specific proton abstraction from one carbon followed by a completely stereospecific protonation at an adjacent carbon by single active site base. We believe that these studies are relevant to a number of basic issues in biochemistry and medicine and that new chemistry and enzymology will result from this work.

此竞争性续约申请旨在继续支持我们的专业 致力于阐明基本的新事物的研究努力 酶和抑制剂作用化学。 在下一个供资期间， 我们会继续在三个主要范畴进行工作： 以及E.大肠杆菌丙酮酸甲酸裂解酶，进一步 干预除卤机理的探讨 催化碳负离子机制的酶和一些 最后对糖苷酶I催化的立体专一性进行了研究。 本课题的研究重点是原核厌氧发酵的关键酶 糖酵解、丙酮酸甲酸裂解酶（PFL）。 最近的工作，包括我们自己的， 提供了暗示性证据，表明反应可能通过 丙酮酸的酶促均裂前所未有。 我们的方法 将涉及PFL模式失活的彻底研究， 次磷酸盐、乙酰次膦酸盐和炔丙基酸。 本人和她姐姐 我们最近发现它是一种非常有效的 酵素 还将研究PFL激活的新模式。 这些研究将需要广泛的努力，需要综合方面的专门知识， 酶纯化，稳定和放射性同位素分析，高场NMR和 EPR研究。 我们最近已经证明，新的醌二甲烷中间体， 通过酶从对卤甲基取代的芳族底物产生 其通过假定的碳负离子机制进行。 这一现象已 为glycoprotein酶I、苯甲酰甲酸脱羧酶和扁桃酸建立 消旋酶 这些研究现在将扩展到黄素依赖性D- 氨基酸氧化酶，并试图确定是否自由基机制 也可以催化这些消除，成为单胺氧化酶。 这项工作是 对药物设计和酶领域具有特别意义 通过醌甲基化物中间体灭活。 最后，我们将进行一些最后的实验， glycoproteinase I具有独特的和进化上重要的能力， 催化从一个碳上提取非立体特异性质子， 通过在相邻碳上的完全立体有择质子化， 活动基地 我们认为，这些研究与一些基本问题有关， 新的化学和酶学将产生 从这项工作中。