MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

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A regulatory motif of fundamental importance to metabolic control is the allosteric modification of enzymatic activity. The long term objective of this application is to increase our understanding of the mechanisms by which allosteric ligands are able to modify enzymatic activity through binding to sites on the enzyme removed from the active site. In particular we are interested in systems in which the allosteric ligands achieve their effects by altering the affinity of enzyme for its substrate. We specifically propose to study three different allosteric systems: prokaryotic phosphofructokinase (PFK) isolated from both Escherichia coli and Bacillus stearothermophilus; carbamoyl phosphate synthetase (CPS) from E. Coli; and NAD-dependent isocitrate dehydrogenase (ICDH) obtained from beef heart mitochondria. All of these enzymes fulfill important regulatory niches in metabolism. Even more importantly for the objectives of this application, however, they present opportunities for answering mechanistic questions that have general relevance to many other allosteric enzymes. By studying these enzymes we hope to be able to answer several questions regarding allosteric response including the following: 1) Is it appropriate to view allosteric behavior in terms of a two-state model even when two X-ray crystal structures are known? 2) Can temperature alter the nature as well as the magnitude of allosteric response? 3) How can two structurally similar ligands have opposite allosteric effects when binding to the same site? and 4) Can a single quantitative model be derived to explain the actions of an allosteric ligand that achieves its effects by altering both the tertiary conformation and the aggregation state of an enzyme? Our approach begins with a systematic and thorough linked-function characterization of the actions of an allosteric ligand. This analysis yields terms that quantify not only the affinity of the substrate and effector ligand, but also the nature and magnitude of the allosteric influence. By monitoring the changes induced in these parameters by modifications of an experimental variable; such as ligand structure, enzyme structure, temperature, enzyme concentration, etc.; insight can be gained into the relationship between the experimental variable and the actions of the allosteric ligand. This approach will be complemented by physical studies; notably fluorescence intensity, spectral distribution, polarization, and lifetime measurements of intrinsic tryptophan residues and covalently attached extrinsic fluorescent probes; to further define the structural consequences resulting from allosteric ligand binding.