This proposal is to continue long-standing studies of kinetic regulatory properties of phosphofructokinase (PFK). PFK is clearly established as the principal rate controlling step in the metabolism of hexose phosphate to pyruvate in all tissues and most organisms. The enzyme represents a striking example of the diversity of regulatory control achieved through complex allosteric and protein-protein interactions. The significance of phosphorylation in the regulation of brain PFK, which contains the unique C isozyme, and in its interaction with actin will be determined. A potential role for phosphorylation in the trafficking of PFK in axons will be studied in vitro and in vivo. In addition, this proposal examines some of the basic mechanisms of allosteric interactions, probing the characteristics of ligand binding sites and the communication of enzyme subunits. The multiple forms of the enzyme with their varying sensitivities to allosteric ligands provide fundamental structural clues to the nature of binding sites. Furthermore, site-directed mutagenesis additional structural correlates of function. The overall objectives are as follows. (a) Chemical modification studies will be coupled with peptide sequencing of catalytic and ligand binding sites of E. coli PFK and the three rabbit isozymes to provide structural information related to the differing sensitivities of these isozymes to allosteric effectors and to define essential features of the sites. These studies will include kinetic, ligand binding, and conformational analysis of the native and modified enzymes. (b) Mutagenesis of E. coli PFK will be carried out with two general goals. The role of specific amino acid residues in the catalytic and allosteric site of the enzymes will be ascertained. Based upon the knowledge gained in the study of the characteristics of the ligand binding sites of the rabbit isozymes, mutations will be designed to modify the specificity of ligand binding sites in the bacterial enzyme. In addition, the communication between subunits will be examined by employing hybrids of native and mutagenized protomers.
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