Energetics of cooperative ligand binding to the active sites of biosynthetic threonine deaminase from Escherichia coli.

Abstract

The sigmoidal kinetics of alpha-ketobutyrate production catalyzed by threonine deaminase are shifted in the presence of the feedback inhibitor isoleucine and the activator valine to control carbon flow through branched-chain amino acid biosynthesis in Escherichia coli. As an initial effort toward developing a molecular mechanism for cooperativity and feedback inhibition in this enzyme, the binding of the substrate analogs 2-aminobutyrate and alanine were measured. Binding isotherms were determined in potassium phosphate at pH 7.5 by detection of an 8-10-fold increase in intrinsic fluorescence of the external aldimine Schiff base of these analogs with the essential pyridoxal phosphate cofactor of the enzyme. Both 2-aminobutyrate and alanine bind cooperatively to four sites on threonine deaminase, with an average dissociation constant of 12.7 and 43.8 mM, respectively. The feedback inhibitor isoleucine decreases the average affinity for the ligands and increases the degree of cooperativity. The activator valine decreases the degree of cooperativity, but gives rise to a slight increase in the average dissociation constant for 2-aminobutyrate and alanine, possibly due to a competitive effect with active site ligands. The temperature dependence of the average affinity of ligands for the active sites indicates that binding is entropically controlled, with average values for delta H0 of +6.0 kcal/mol for alanine and +4.87 kcal/mol for 2-aminobutyrate. Since none of the ligands under investigation had any effect on the tetrameric quaternary structure of the enzyme as judged by sedimentation equilibrium, an initial attempt to describe cooperative ligand association with threonine deaminase was undertaken by analysis of the binding isotherms in terms of a two-state model in which the enzyme equilibrates between two conformations, T and R, that differ in their affinity for ligands. This analysis suggests that cooperative binding to the enzyme is largely the result of the high preferential affinity of ligands for the R state. In addition, estimates for the allosteric equilibrium constant L range from 2 to 5, in the absence of modifiers, to 35 in the presence of isoleucine, and 0.9 in the presence of valine. To a first approximation, the data are consistent with isoleucine and valine regulating threonine deaminase by shifting the allosteric equilibrium between the different affinity forms.