HEAVY ATOM ISOTOPE EFFECTS ON ENZYMATIC REACTIONS

正在使用重原子同位素效应和相关的动力学技术 研究多种酶的作用机制。特别的 兴趣在于动力学机制、化学机制和催化作用。 机制。这些研究利用了x射线的最新进展。 结晶学、定点突变和各种其他 技巧。到目前为止对天冬氨酸转氨酶的研究揭示了 除了众所周知的T-R之外，还有丰富的构象变化 过渡。对突变酶的研究尤其具有启发性。 将进行进一步研究，以了解各种不同的 作为催化机制一部分的构象变化， T和R状态催化的性质，官能团在 催化作用和pH依赖的动力学机制。要实现 这，碳、氮和氢的同位素效应将被测量 天然酶、催化亚单位和各种突变酶。 核酮糖二磷酸羧化酶/加氧酶是一种重要的 典型的羧基酶，尤其神秘，因为 与羧化作用竞争的简单的氧化作用。碳和碳的研究 氧同位素对天然酶和突变酶的影响将用于 了解羧化步骤是否可逆，是否控制 底物构象是CO2/O2专一性的一个重要方面， 过渡态结构是恒定的还是可变的 酶的形式，无论进入二氧化碳与金属在 羧化步骤，以及是否有可能增加CO2/O2 酶的专一性。磷脂酶A2的研究将被用于 了解酶反应的机理，在 具有酶结构的过渡态结构和动力学 胶束和囊泡中的酶催化作用。胶束中的研究和 囊泡将被用来将同位素效应研究转移到 多相反应，这是传统动力学研究的一个领域 用处有限。

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.