This is a proposal to continue long-standing studies of structure/function relationships with respect to the catalytic and regulatory properties of phosphofructo-1-kinase (PFK). PFK is clearly established as the major rate controlling step in the metabolism of hexose phosphate to pyruvate in all mammalian tissues and almost all other organisms. A detailed study of the nature of the ligand binding sites and of the conformational changes associated with allosteric control is important to our understanding of the regulation of carbohydrate metabolism in normal and pathological states. The principal goal will be the detailed analysis of the regulatory properties of the recently cloned and expressed PFK (C isozyme) from rabbit brain. Critical residues involved in ligand binding and in the conformational change that transmits the allosteric signal will be identified using site-directed mutagenesis of residues that are thought to comprise each of the four binding sites for organic regulatory ligands. To distinguish between those mutations that prevent ligand binding and those that interfere with conformational changes, kinetic studies of the mutants will be supplemented by studies of equilibrium binding of regulatory ligands and by protein conformational analysis, examining the effects of ligands on the rate of thiol reactivity, the rate of limited proteolysis, and changes in intrinsic fluorescence. In addition, the genomic structure of rabbit PFK C isozyme will be examined to determine intron-exon junctions and the structural features of the 5' flanking region. Finally, site-directed mutagenesis studies of the role of specific amino acid residues in the interaction of phosphoryl donors of ATP-dependent PFK from Escherichia coli and the PPi-dependent PFK from Propionibacterium freudenriechii will be completed.
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