HEAVY ATOM ISOTOPE EFFECTS ON ENZYMATIC REACTIONS

目前正在使用重原子同位素效应和相关的动力学技术

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.