HEAVY ATOM ISOTOPE EFFECTS ON ENZYMATIC REACTIONS

重原子同位素对酶反应的影响

• 批准号: 2181766
• 负责人: MARION H OLEARY
• 金额: $ 16.62万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-06-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181766
• 关键词: aspartate carbamoyltransferase; carbon; carbon carbon lyase; carbon dioxide; catalyst; chemical kinetics; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; hydrogen; micelles; mutant; nitrogen; nonradiation isotope effect; phospholipase A2; stable isotope

Heavy-atom isotope effects and related kinetic techniques are being used to study the mechanisms of action of a variety of enzymes. Of particular interest are kinetic mechanism, chemical mechanism, and catalytic mechanism. These studies take advantage of recent advances in x-ray crystallography, site-directed mutagenesis, and a variety of other techniques. Studies of aspartate transcarbamylase to date have revealed a wealth of conformational changes in addition to the well-known T-R transition. Work with mutant enzymes has been particularly revealing. Further studies will be conducted in order to learn about the various conformational changes that are part of the catalytic mechanism, the nature of catalysis by T and R states, the roles of functional groups in catalysis, and the pH dependence of the kinetic mechanism. To achieve this, carbon, nitrogen, and hydrogen isotope effects will be measured for the native enzyme, the catalytic subunit, and various mutant enzymes. Ribulose bisphosphate carboxylase/oxygenase is an important and prototypical carboxylase that is especially enigmatic because of the facile oxygenation that competes with carboxylation. Studies of carbon and oxygen isotope effects with native and mutant enzymes will be used to learn whether the carboxylation step is reversible, whether control of substrate conformation is an important aspect of CO2/O2 specificity, whether transition-state structure is constant or variable for various forms of the enzyme, whether entering CO2 interacts with the metal during the carboxylation step, and whether it is possible to increase the CO2/O2 specificity of the enzyme. Studies of phospholipase A2 will be used to learn about the mechanism of the enzymatic reaction, variations in transition-state structure with enzyme structure, and dynamics of enzymatic catalysis in micelles and vesicles. Studies in micelles and vesicles will be used to move isotope-effect studies into the area of heterogeneous reactions, an area in which traditional kinetic studies have been of limited usefulness.

正在使用重原子同位素效应和相关的动力学技术 研究各种酶的作用机制。特别 主要有动力学机理、化学机理和催化机理 机制这些研究利用了X射线的最新进展 晶体学、定点诱变和各种其他方法， 技术. 迄今为止，天冬氨酸转氨甲酰酶的研究表明， 除了众所周知的T-R， 过渡对突变酶的研究尤其具有启发性。 将进行进一步研究，以了解各种 构象变化是催化机制的一部分， T和R态催化的性质，官能团在催化中的作用， 催化，和pH依赖性的动力学机制。实现 碳、氮和氢同位素效应将被测量， 天然酶、催化亚基和各种突变酶。 核酮糖二磷酸羧化酶/加氧酶是一种重要的 一种典型的羧化酶，由于其 与羧化作用竞争的易氧化作用。研究碳和 天然和突变酶的氧同位素效应将用于 了解羧化步骤是否可逆， 底物构象是CO2/O2特异性重要方面， 过渡态结构对于各种情况是恒定的还是可变的 酶的形式，是否进入CO2与金属相互作用， 羧化步骤，以及是否可能增加CO2/O2 酶的特异性。磷脂酶A2的研究将用于 了解酶促反应的机制， 过渡态结构与酶的结构， 胶束和囊泡中的酶催化。胶束研究和 囊泡将用于将同位素效应研究转移到 非均相反应，传统的动力学研究领域， 用处有限。