Slow conformational changes, the interaction of domains within a subunit of an enzyme, and the interaction between subunits of an oligomeric enzyme are important for the function and regulation of many enzymes, but the dynamics of these processes are not fundamentally well understood. The goal of this proposal is to utilize rat liver glucokinase and yeast hexokinase as well-defined model systems to obtain quantitative structural and dynamic information in order to define the possible functional significance of these physical properties. These structural dynamics will be related to the physiological control of these enzymes, particularly of glucokinase, a key regulatory enzyme in liver. We propose to define the structural basis for the positive cooperativity that glucokinase displays with glucose and to test whether glucokinase undergoes a change in domain interactions subsequent to sugar binding, similar to that of hexokinase; the specificity of the conformational changes will be determined from kinetics and from fluorescent probes. We will characterize the structural basis for the palmitoyl CoA inhibition, sulfhydryl oxidation, and liver-derived factor stabilization of glucokinase with ligand binding, spectroscopic, and structural studies. The physiological significance of these proposed regulatory properties will be determined by correlation with changes in metabolism of primary hepatocyte cultures. The changes induced in hexokinase upon sugar and/or nucleotide binding will be further defined by determining the contribution of individual amino acids to the altered kinetic and physical properties of yeast hexokinase mutants, by 13-C NMR distance measurements of cleft closure, and by comparison to the known three dimensional structure. The unique aspects of these studies are: (a) the focus on hysteretic aspects of enzyme regulation, (b) the investigation of domain interactions in a well defined structure, (c) the structural comparisons between a well characterized yeast enzyme and its related mammalian counterpart, and (d) the physiological comparisons to validate the regulatory mechanisms under investigation. These studies will provide a better understanding of the role of hysteresis and domain interactions in the molecular and cellular properties of regulatory enzymes, in general, and of the metabolic disorders of glucose phosphorylation in diabetes.
