STRUCTURE AND REGULATION OF A PROTEIN KINASE/PHOSPHATASE

In biological systems, most processes are under the control of a variety of regulatory mechanisms. For example, the glyoxylate bypass of Escherichia coli is controlled by the phosphorylation of isocitrate dehydrogenase (IDH), by regulation of the expression of the glyoxylate bypass operon and by allosteric mechanisms. Our long term goal is to characterize the functioning and coordination of these regulatory mechanisms. This proposal focuses on the structure/function relationships of IDH kinase and IDH phosphatase and the regulation of the phosphorylation cycle in vivo. IDH kinase and phosphatase have been found to be physically associated, probably on the same polypeptide chain. In an effort to determine the regulatory and structural significance of this bifunctional structure, the relationships between the functional domains will be characterized. In addition to the methods of protein chemistry, this study will make extensive use of recombinant DNA technology, including mutagenesis of the gene encoding IDH kinase/phosphatase. Specific problems to be addressed include the location of these domains, the degree to which they are physically autonomous and the potential for interdomain allosteric communication. During the analysis of the structure of IDH kinase/phosphatase, a variety of normal and mutant clones of the gene encoding this protein are being constructed. These clones provide an ideal approach to the study of the IDH phosphorylation cycle in vivo. The cellular level and properties of IDH kinase/phosphatase will be manipulated using these clones and the effects of these perturbations will be characterized. The results of these experiments will provide the basis for the construction of models which describe the regulation of IDH phosphorylation in the intact organism.