The long-term objective is understanding mechanisms of control of the catalytic activity of a key enzyme in a signal transduction pathway which modulates gene expression in response to carbon source availability. 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. GK is inhibited by binding of dephospho-IIIglc of the phosphotransferase system and by fructose 1,6-bisphosphate (FBP). The goals of this research are to understand the interactions by which IIIglc recognizes one of its cognate target proteins, to elucidate the roles of cation promoted association (CPA) by Zn(II) in the recognition, to evaluate the coupling of local and long-range conformational changes in GK to IIIglc binding, to test models for IIIglc inhibition, to define the role of the GK dimer-tetramer assembly in FBP inhibition, and to evaluate the physiological roles and significance of the regulation by IIIglc, FBP and CPA.
These aims will be achieved by characterizing the energetics of IIIglc binding to wild type and specific mutant GKs, construction and characterization of specific GK mutants to test roles of particular interactions, and quantitation of the assembly reaction for wild type GK and selected mutants. Roles of particular regions in the conformational transitions associated with IIIglc inhibition will be assessed by constructing His2 transition metal binding sites that will allow metal binding to mimic IIIglc effects. Mutants which specifically lack one of the regulatory properties but retain normal catalytic and other regulatory functions will be crossed back into the chromosome by using bacterial genetics methods. Evaluation of the effects of the specific mutations on glucose control of glycerol metabolism and expression of the glp regulon elements in vivo will indicate the physiological role of each of the types of regulation. A novel role for Zn(II), already widely implicated in normal and pathological biological processes, will be characterized. The energetic and structural properties of a defined coil-helix transition, implicated in hormone binding and transcription factor associations, will be defined and its role in protein-protein interactions elucidated. The interaction between GK and IIIglc involves only two salt bridges which have different degrees of solvent exposure; the contributions of these different types of salt bridges to protein-protein interactions will be defined. Finally, the small number of amino acids in the GK-IIIglc complex will permit rigorous detailed characterization of this protein-protein interaction.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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Texas Agrilife Research
Schools of Earth Sciences/Natur
College Station
United States
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