Cooperative binding of the feedback modifiers isoleucine and valine to biosynthetic threonine deaminase from Escherichia coli.

Control of the regulatory enzyme threonine deaminase from Escherichia coli is achieved by isoleucine inhibition and valine activation. The mechanism by which these heterotropic effectors regulate the enzyme was investigated by measuring the binding of isoleucine and valine by spectroscopic, kinetic, calorimetric and equilibrium dialysis techniques. The addition of isoleucine or valine to threonine deaminase resulted in large changes in the intrinsic fluorescence of the two tryptophans per polypeptide chain. Slightly cooperative binding isotherms for isoleucine were obtained in potassium phosphate, pH 7.5, yielding an average dissociation constant of 4.91 microM, which was confirmed by equilibrium dialysis measurements. Valine binding was much more cooperative, and yielded an average dissociation constant of 122 microM. Titration calorimetry experiments indicated that cooperative heterotropic ligand binding was exothermic, and yielded a stoichiometry of four isoleucine bound per tetrameric enzyme, with an average enthalpy of -10.70 kcal/mol. Valine also bound to four sites per tetramer, with an average enthalpy of -7.45 kcal/mol. The effect of ligands on the fluorescence and circular dichroism spectra of the essential pyridoxal phosphate cofactor indicates that isoleucine and valine bind to effector sites that are distinct from the active sites in threonine deaminase. Shifts in the kinetic properties of threonine deaminase promoted by isoleucine and valine binding are to a first approximation consistent with analyses of effector binding isotherms in terms of a simple two-state model, and suggest that isoleucine regulates threonine deaminase by preferentially binding to the low activity T state, whereas valine binds preferentially to the high activity R state. Finally, analyses of heterotropic effector binding isotherms suggest that active site ligands may have significant affinity for the regulatory sites, which gives rise to underestimates for the allosteric equilibrium constants determined from substrate analog binding isotherms.

Duke Scholars

Author Eric Leo Eisenstein Medicine, Cardiology