AN EXPANDED 2-STATE MODEL ACCOUNTS FOR HOMOTROPIC COOPERATIVITY IN BIOSYNTHETIC THREONINE DEAMINASE FROM ESCHERICHIA-COLI

The linkage between substrate and regulatory effector binding to separ ate sites on allosteric enzymes results in shifts in their sigmoidal k inetics to regulate metabolism, Control of branched chain amino acid b iosynthesis in Escherichia coli occurs in part through shifts in the s igmoidal dependence of alpha-ketobutyrate production promoted by isole ucine and valine binding to biosynthetic threonine deaminase. The stru ctural similarity of threonine, valine, and isoleucine have given rise to suggestions that there may be competition among different ligands for the same sites on this tetrameric enzyme, resulting in a complex p attern of regulation. In an effort to provide a coherent interpretatio n of the cooperative association of ligands to the active sites and to the effector sites of threonine deaminase, binding studies using sing le amino acid variants were undertaken, A previously-isolated, feedbac k-resistant mutant identified in Salmonella typhimurium, ilvA219, has been cloned and sequenced, The phenotype is attributable to a single a mino acid substitution in the regulatory domain of the enzyme in which leucine at position 447 is substituted with phenylalanine, The mutant exhibits hyperbolic saturation curves in both ligand binding and stea dy-state kinetics. These results, in addition to calorimetric and spec troscopic measurements of isoleucine and valine binding, indicate that the low affinity (T) state is destabilized in the mutant and that it exists predominantly in the high affinity (R) conformation in the abse nce of ligands, providing an explanation for its resistance to isoleuc ine. Chemical and spectroscopic analyses of another mutant, in which a lanine has replaced an essential lysine at position 62 that forms a Sc hiff base with pyridoxal phosphate, indicate that the cofactor is comp lexed to exogenous threonine and is therefore unable to bind additiona l amino acids at the active sites. Isoleucine and valine binding to th is inactive, active site-saturated enzyme revealed that it too was sta bilized in the R state, yielding binding constants in excellent agreem ent with the leucine to phenylalanine mutant, The lysine to alanine mu tant was further utilized to demonstrate that both threonine and 2-ami nobutyrate bind with stronger affinity to the regulatory sites than to the active sites, A direct consequence of these results is that subst rates and analogs have a synergistic effect on the allosteric transiti on since, in effect, they act as both homotropic and heterotropic effe cters. When these coupled equilibria are considered in an expanded two -state model, good agreement was obtained for the allosteric parameter s determined from homotropic and heterotropic ligand binding, and furt hermore, they provide an estimate of 4.3 kcal/mol for the average ener getic difference between the T and R conformations.