The goal of this research is to understand the molecular basis for regulation of glycolytic/gluconeogenic pathway flux in liver by focusing on the enzymes of the Fru 6-P/Fru 1,6-P2 substrate cycle, including 6-phosphofructo-l-kinase (6PFlK), fructose-1,6- bisphosphatase (Fru-1,6-P2ase), and 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P2ase). The major emphasis of the proposed work will be on 6PF2K/Fru 2,6-P2ase. We have put forth the hypothesis that this bifunctional enzyme consists of two independent domains. Site-directed mutagenesis will be employed to test this hypothesis and to determine the molecular basis for catalysis and regulation of the enzymes, reactions. If each domain can be expressed separately in a heterologous expression system, with retention of its predicted activity, the hypothesis will be substantiated. The properties of the separate kinase and bisphosphatase domains will also be compared with those of the native enzyme to elucidate the advantages of enzyme functionality. The nature of the reciprocal regulation of the two activities by cAMP-dependent phosphorylation will be studied with mutant forms of the enzyme where the phosphorylation site has been altered. The evolutionary relationship between the bisphosphatase domain and phosphoglycerate mutase, in particular the conservation of the mutase active site residues in the bisphosphatase, provides a basis for the site- directed mutagenesis at the bisphosphatase active site. Putative active site residues will be systematically altered in order to identify the critical residues for catalysis in both the kinase and bisphosphatase reactions. Site-directed mutagenesis will also be employed to identify the Fru 2,6-P2 binding site in 6PF1K and Fru- 1,6-P2ase and to determine what role phosphorylation has in their regulation. Hormonal control of gene expression of 6PF2K/Fru-2,6- P2ase, 6PF1K, and Fru-1,6-P2ase will also be characterized, the mechanism of these effects elucidated, and the relationship of this long-term regulation to overall control of gluconeogenesis/glycolysis in various dietary and hormonal states evaluated. If it is determined that control of the amount of these enzymes is at the level of transcription then their genomic structures will be determined, including the location and sequence of putative hormone response element knowledge gained from the above studies will allow expression of wild-type and mutant enzyme forms in liver cell lines in the future and permit the effects of such changes to be tested on function and control of the entire pathway. Elucidation of the molecular mechanisms whereby hormones regulate glycolytic/gluconeogenic flux should lead to new approaches for treatment of disease states where there are derangements in control of these pathways, such as diabetes.
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