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  • Title: Allosteric regulation of biosynthetic threonine deaminase from Escherichia coli: effects of isoleucine and valine on active-site ligand binding and catalysis.
    Author: Eisenstein E.
    Journal: Arch Biochem Biophys; 1995 Jan 10; 316(1):311-8. PubMed ID: 7840631.
    Abstract:
    The sigmoidal steady-state kinetics of biosynthetic threonine deaminase from Escherichia coli is allosterically controlled by isoleucine and valine, the end-products of branched-chain amino acid biosynthesis. A basis for the regulation of threonine deaminase by heterotropic effectors has been studied by capitalizing on the intrinsic fluorescence of the essential pyridoxal phosphate cofactor in this enzyme in kinetic and equilibrium binding studies with the substrate analog D-threonine. D-Threonine binds cooperatively to four sites on the free enzyme, with an average dissociation constant of 19.8 mM. However, in the presence of saturating valine, or isoleucine, the D-threonine binding isotherms are noncooperative and characterized by dissociation constants of 3.9 and 24.8 mM, respectively. The rate of association of D-threonine with threonine deaminase in the presence of the regulatory ligands was biphasic. Analysis of the data in terms of a two-step scheme whereby the internal aldimine Schiff base in the initial encounter complex undergoes transimination with D-threonine to form an external aldimine yielded estimates for overall binding constants that were in good agreement with those determined from equilibrium binding isotherms. These analyses indicate that the positive allosteric effector valine acts solely to alter the binding of D-threonine to the active sites of threonine deaminase by shifting the equilibrium between a low-affinity and high-affinity state, consistent with predictions from a simple two-state model. However, isoleucine has a compound effect on the enzyme. The negative allosteric ligand promotes decreases in the rate and equilibrium constants for encounter complex formation, consistent with its preferential binding to the low-affinity state of the enzyme. In addition, however, isoleucine promotes a decrease in the transimination rate and equilibrium constants. Since transimination is generally considered to be protein-catalyzed in pyridoxal phosphate requiring enzymes, it is proposed that isoleucine affects both binding and catalysis in threonine deaminase, which provides a possible explanation for the inadequacy of a simple two-state model to describe the allosteric regulation of this enzyme.
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