AMINO-ACID SUBSTITUTIONS IN THE C-TERMINAL REGULATORY DOMAIN DISRUPT ALLOSTERIC EFFECTOR-BINDING TO BIOSYNTHETIC THREONINE DEAMINASE FROM ESCHERICHIA-COLI

Shifts in the sigmoidal kinetics of allosteric threonine deaminase pro moted by isoleucine and valine binding control branched chain amino ac id biosynthesis in Escherichia coli. A highly conserved alpha-helix in the C-terminal regulatory domain of the tetrameric enzyme was previou sly implicated in effector binding and feedback inhibition. Double (44 7, 451) and triple (447, 451, 454) alanine replacements for the conser ved amino acids leucine 447, leucine 451, and leucine 454 in this regi on yield enzyme variants that show increased sigmoidality in steady-st ate kinetics, and which are less sensitive to the allosteric modifiers isoleucine and valine, Equilibrium binding studies using fluorescence , enzyme kinetic, and calorimetric approaches indicate that the enzyme variants possess reduced affinity for isoleucine and valine, and sugg est that heterotropic ligands can bind to the same site to promote the ir different effects. The increase in sigmoidal kinetics for the mutan ts relative to wildtype threonine deaminase may be attributable to the elimination of L-threonine binding to the effector sites, which activ ates the wild-type enzyme, Enzyme kinetic data and isotherms for activ e site ligand binding to the mutants can be analyzed in terms of a sim ple two-state model to yield values for allosteric parameters that are consistent with previous estimates based on an expanded two-state mod el for homotropic cooperativity for threonine deaminase.