This project explores the relationship betweeen the catalytic activities and the physical, chemical, and structural properties of enzymes. Four aspects of enzyme catalysis will be studied: (1) The nature of the weak and strong chemical bonding interactions which catalyze covalent transformations in the reacting substrate. (2) The influence of allosteric effectors on the activation energies of enzyme-catalyzed processes. (3) The interrelation between enzyme conformational change and catalysis. (4) Metabolite transfer mechanistic in bienzyme complexes which catalyze sequential steps in a metabolic pathway. To investigate these phenomena, four enzyme systems are selected for detailed mechanistic studies: (1) the liver alcohol dehydrogenases from human and horse liver, (2) the bienzyme, PLP-requiring tryptophan synthase from E. coli, (3) the PLP requiring cystathionine gamma-synthase from E. coli, and (4) aldehyde dehydrogenase from sheep liver. The project emphasizes the use of rapid kinetic techniques (especially rapid scanning and single wavelength stopped-flow UV-visible spectroscopy) to study these systems. Other techniques of importance to the project include stopped-flow fluorescence spectroscopy 1H and 31P FT NMR, UV-visible microspectrophotometry of enzyme crystals and chemical modification of enzyme side chain residues. These techniques will be used to detect and identify chemical intermediates in catalysis, to search for conformational changes linked to catalysis, and to examine the influence of allosteric effectors on the interconversion of chemical intermediates on the catalytic path. These studies are expected to significantly advance our understanding of some of the least well understood aspects of enzyme catalysis.
