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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Laughlin, Maren R
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Rosalind Franklin University
Schools of Medicine
North Chicago
United States
Zip Code
Kemp, Robert G; Gunasekera, Dhammika (2002) Evolution of the allosteric ligand sites of mammalian phosphofructo-1-kinase. Biochemistry 41:9426-30
Chang, Simon H; Kemp, Robert G (2002) Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase. Biochem Biophys Res Commun 290:670-5
Wang, X; Kemp, R G (2001) Reaction path of phosphofructo-1-kinase is altered by mutagenesis and alternative substrates. Biochemistry 40:3938-42
Gunasekera, D; Kemp, R G (2000) Genomic organization, 5'flanking region and tissue-specific expression of mouse phosphofructokinase C gene. Gene 260:103-12
Deng, Z; Wang, X; Kemp, R G (2000) Site-directed mutagenesis of the fructose 6-phosphate binding site of the pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica. Arch Biochem Biophys 380:56-62
Wang, X; Kemp, R G (1999) Identification of residues of Escherichia coli phosphofructokinase that contribute to nucleotide binding and specificity. Biochemistry 38:4313-8
Deng, Z; Roberts, D; Wang, X et al. (1999) Expression, characterization, and crystallization of the pyrophosphate-dependent phosphofructo-1-kinase of Borrelia burgdorferi. Arch Biochem Biophys 371:326-31
Li, Y; Rivera, D; Ru, W et al. (1999) Identification of allosteric sites in rabbit phosphofructo-1-kinase. Biochemistry 38:16407-12
Gunasekera, D; Kemp, R G (1999) Cloning, sequencing, expression, and purification of the C isozyme of mouse phosphofructokinase. Protein Expr Purif 16:448-53
Wang, X; Deng, Z; Kemp, R G (1998) An essential methionine residue involved in substrate binding by phosphofructokinases. Biochem Biophys Res Commun 250:466-8

Showing the most recent 10 out of 32 publications