The Molecular Mechanisms Governing the Catalysis of 6-Phosphofructo-2-kinase

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The liver bifunctional enzyme, 6-phosphofructo-2-kinase fructose- 2,6-bisphosphatase has been isolated and characterized by a number of biophysical and biochemical methods. The PI has determined its protein and cDNA sequence and has shown that the enzyme is composed of two independent domains: a NH2-terminal kinase domain and a COOH-terminal bisphosphatase domain. The latter is structurally and functionally homologous to the phosphoglycerate mutase/acid phosphatase family. Recent structural analysis of the 2-kinase domain has revealed a structural homology with 6-phosphofructo-1-kinases. It is planned to use structural information and biochemical approaches as well as methods of site specific mutagenesis to extend knowledge of the mechanisms involved in 6PF-2-K catalysis. Four questions will be studied: 1) Based on structural information to employ site directed mutagenesis at the site of the hypothesized nucleotide binding fold of 6-phosphofructo-2-kinase in order to confirm its role in ATP binding. 2) To determine the role of several; cysteinyl residues which have been implicated from protein modification experiments as being important in the 2-kinase reaction. 3) To determine the anomeric specificity of the 6-PF-2-K reaction. 4) Based on comparison of the topological structure of 6PF- 1-K and that predicted for 6PF-2-K to alter residues responsible for Fru 6-P binding. The effects of substitutions will be studied by measuring aspects of enzyme activity, Fru 6-P and ATP affinity, spectral properties, and protein structure.