A regulatory motif 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. Three different allosteric enzymes will be studied as model systems: phosphofructokinase (PFK) from E. coli, PFK from B. stearothermophilus; and carbamoyl phosphate synthetase (CPS) from E. coli.
In Specific Aim I, a series of mutant hybrids of PFK from both E. coli and B. stearothermophilus will be constructed in which the binding sites on 3 of the subunits will have been knocked out with mutations of the positively charged residues that line the binding sites. The remaining wild-type subunit will create a single high-affinity substrate binding site and a single high affinity allosteric binding site. By varying which residues are mutated, each of the 4 site-site interactions will be isolated and studied individually.
Specific Aim II entails a systematic evaluation of the relationship between allosteric action and binding specificity. Substrate and allosteric ligand analogs of CPS and PFK from both E. coli and B. stearothermophilus will be evaluated to test our hypothesis that allosteric ligands can have an inverted effect on binding specificity if the coupling free energy that establishes the allosteric effect is entropy-dominated.
In Specific Aim III we will evaluate how the equilibrium effects of allosteric ligands are manifested in kinetics of ligand binding and dissociation in order to paint a more accurate picture of the actually perturbation that is involved. Particular attention will be paid to those instances where the substrate and the allosteric ligand appear to bind independently but where there is evidence from the underlying thermodynamics that the sites may be 'silently' coupled. These investigations should provide significant insight into the specific molecular mechanisms by which allosteric ligands achieve their effects. This information will ultimately prove useful in medical efforts, such as drug design, which require a precis knowledge of the structural consequences of ligand binding and of the properties that underly the specificity of protein-ligand interactions.
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