A regulatory motive of fundamental importance to metabolic control is the allosteric modification of enzymatic activity by metabolites. The long-term goal of this research program is to understand the molecular basis for allosteric regulation of enzyme activity. In particular we are interested in systems in which the allosteric ligands achieve their effects by altering the affinity of the enzyme for its substrate. Phosphofructokinase(PFK) from E. coli and B. stearothermophilus will be investigated as model systems. These enzymes are homotetramers containing a single active site and a single allosteric site per subunit. Despite this relatively simple composition, 10 unique pair-wise allosteric interactions can be identified. Hybrid forms of these enzymes have been produced that isolate individual allosteric interactions that collectively act to produce the regulatory properties of the tetramic enzyme. We propose to investigate the molecular basis for the allosteric properties displayed by each of these hybrids because of the defined nature of the functional interaction in each case. Improvements in the procedures used to produce these hybrids will be developed by creating an expression strain of E. coil that does not have either gene for PFK and by refining a procedure for producing hybrids in vivo by co-expressing both parent proteins in the same cell. A strategy is proposed for isolating each 2:2 hybrid that will allow for the isolation of each unique homotropic interaction and for the demonstration of how multiple interactions combine within a dimeric theoretical framework. The putative conduit by which allosteric influence is transmitted in each hybrid displaying a single interaction will be located through the systematic use of site-directed mutagenesis. Concurrently the structural basis for the allosteric communication will be studied with X-ray crystallography and time-resolved fluorescence spectroscopy. The latter technique should produce information regarding the dynamic properties of the protein that will complement the structural information obtained from crystallography. Finally PFK from both E. coil and B. stearothermophilus will be carefully compared with respect to how substrate and allosteric ligand analogs trigger their allosteric behavior. These enzymes differ in the apparent thermodynamic basis for their allosteric properties, a difference that might be related to differences in binding specificity.
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