Mechanisms of Allosteric Influence on Enzymes Activity

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A regulatory motif of fundamental importance to metabolic control, and increasingly to drug design, is the allosteric modification of enzyme activity. The long-term goal of this research program is to understand the molecular basis for allosteric regulation. Currently we are focused on systems in which allosteric ligands achieve their effects by altering the affinity of the enzyme for its substrate. Phosphofructokinase (PFK) from a variety of bacterial sources will be investigated as model systems and as a prelude to eventual studies of eukaryotic forms of the enzyme. These enzymes are homotetramers containing a single active site and a single allsoteric site per subunit. Despite this relatively simple composition, 10 unique pair-wise allosteric interactions can potentially exist. Hybrid forms of these enzmes have been produced that isolate individual allosteric interactions, the sum of which quantitatively explain the allosteric response in the native tetramer. We propose to address four questions of broad relevance to many allosteric enzymes. The first question is whether the different heterotropic energetic interactions identified in the previous grant term arise from quasi- independent interaction pathways within the tetramer. We will address this question by mapping the pathways through the use of point mutations introduced selectively into the hybrids and assessing the independence of the energetic perturbation on individual pathways. The second question is whether the entropy change that contributes to the action of an allosteric ligand is related to changes in enzyme dynamics. This question will be approached by creating hybrids with a single allosteric interaction and a single tryptophan located in a known position relative to the interacting sites. The tryptophan will serve as a reporter for time-resolved fluorescence experiments designed to reveal its local degree of mobility. By constructing a library of hybrids, each with a tryptophan in a different position, a comprehensive assessment of structural dynamics changes throughout a single subunit should be possible. These studies will be complemented by analogous methyl-TROSY NMR experiments measuring the side-chain dynamics of leucine, isoleucine, and valine residues. The third question relates to whether limiting structural forms of an enzyme, obtained when either allosteric inhibitors or substrates and activators bind, reveal the structural conflict that causes the antagonism between the binding of inhibitor and the binding of substrate. The quaternary shift that occurs when an inhibitor binds to prokaryotic PFK exemplifies such a major conformational change, and it will be studied with a combination of mutagenesis, X-ray crystallography, F¿rster Resonance Energy Transfer, and NMR approaches. Finally, the structural features of the allosteric ligand that are important to establishing the nature and magnitude of the allosteric response will be investigated by systematically characterizing various allosteric ligand analogs. It is necessary to understand whether these are separable features of ligand structure if one is to eventually design drugs that target allosteric sites.