The long-term objective is understanding mechanisms of control of the catalytic activity of the key enzyme in a signal transduction pathway which modulates gene expression in response to the availability of carbon sources. The enzyme is Escherichia coli glycerol kinase (GK), which catalyzes the MgATP-dependent phosphorylation of glycerol to sn-glycerol- 3-phosphate, which is the inducer for the elements of the glp regulon. Its catalytic activity is inhibited by the binding of fructose 1,6- bisphosphate (FBP) or dephospho-enzyme lllglc of the phosphoenolpyruvate:sugar phosphotransferase system, and the kinetics with respect to ATP show apparent negative cooperativity. The inhibition by lllglc is relieved by reversible phosphorylation of lllglc. the regulatory properties are physiologically-relevant and result in efficient inducer synthesis if glucose becomes unavailable while glycerol is available. Conversely, if glucose again becomes available, inducer synthesis is shutdown by V-system controls which operate even in the presence of high glycerol concentrations. The regulatory behavior will be rigorously characterized by using kinetics, ligand binding, and spectroscopic methods.
The specific aims are to characterize the mechanisms and energetics of the regulation and the mechanism by which phosphorylation of lllglc relieves its inhibition. both wild type GK and lllglc will be characterized, as well as mutants of each enzyme which have been shown to affect FBP or lllglc regulation. The availability of the crystal structures of GK and the GK-lllglc complex greatly facilitates interpretation of the results, and additional mutants may be constructed to test hypotheses that develop from further refinement of the structures. Possible effects of regulation of GK activity on its subcellular localization will be studied by using electron microscopy and immunolocalization. Regulatory properties of other members of a widely- distributed family of bacterial sugar kinases that have primary structures that are similar to GK will be compared to those of GK to investigate relations between enzyme structure and regulation and control of gene expression. The results will provide insights into mechanisms of the control of gene expression in living systems.
|Feese, M; Pettigrew, D W; Meadow, N D et al. (1994) Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation. Proc Natl Acad Sci U S A 91:3544-8|
|Liu, W Z; Faber, R; Feese, M et al. (1994) Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc. Biochemistry 33:10120-6|