The structural basis for the enzymatic activity and allosteric regulation of Eschericia coli glycerol kinase, which is an essential regulatory enzyme for glycerol metabolism in all organisms examined, will be probed by a combination of X-ray crystallographic studies, site-directed and random mutagenesis, reaction kinetic analysis and synthesis of novel transition- state and bisubstrate compounds. Glycerol kinase, which catalyzed the MgATP dependent phosphorylation of glycerol to form glycerol-3-phosphate, is a rare example of a strictly velocity modulated enzyme, suggesting that the regulatory behavior is a property of the transition state of the reaction. It also displays negative cooperativity with respect to ATP and half-site reactivity with respect to all substrates. However, a unique feature of glycerol kinase is that it is regulated by two different allosteric effectors in an additive manner: a product of glycolysis (fructose 1,6-bisphosphate) and signal transducing protein, IIIglc, depending on the state of phosphorylation of the regulatory protein. As crystals and three-dimensional structures have been obtained of the regulatory protein alone, the enzyme alone, and the complex of the two proteins together, the proposed research is uniquely poised to provide the first detailed picture of a phosphorylation-dependent protein-protein regulatory interaction. Mutants of glycerol kinase individually defective in each of the three aspects of the allosteric response have been prepared, and their structures will be analyzed to ascertain the structural consequences of the mutations. The long term goals are to determine the basis for the phosphoryl transfer reaction, and how it is controlled by the oligomeric states of the proteins and how allosteric effectors regulate the enzymatic activity. The enzyme will also be used as a system for development of potential transition-state analogs, which are not at present available for phosphoryl transfer enzymes. The project is expected to shed light on the general nature of intracellular communication, which is primarily effected by phosphoryl transfer reactions.
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