A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2,[69][70][71][72][73][74][75][76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D., and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition.Biosynthetic threonine deaminase (EC 4.2.1.16; TD) 1 is a pyridoxal phosphate enzyme that catalyzes the deamination of threonine into R-ketobutyrate.Allosteric regulation of bacterial TD by its effectors isoleucine and valine was studied initially in the pioneer works of Umbarger (1) and Monod et al. (2). The kinetics of the reaction were further investigated extensively using an overproduced recombinant Escherichia coli enzyme (3-9). E. coli TD exhibits sigmoidal substrate saturation behavior in the absence of isoleucine. The feedback inhibitor isoleucine decreases the affinity for threonine and increases the cooperativity of the kinetics. On the other hand, valine increases the affinity for threonine, decreases the degree of cooperativity of the kinetics, and reverses the inhibition by isoleucine.The kinetics of the reaction catalyzed by TD have been studied for various species, including bacteria (10-12), yeast (13), and plants (14-16). The effect of isoleucine and valine on these enzymes was shown to be similar to that described for the E. coli enzyme. However, some differences exist at the level of enzyme kinetics and the allosteric effect of isoleucine. Indeed, in the absence of isoleucine, the yeast (13) enzyme exhibits sigmoidal kinetics, similar to those of the E. coli enzyme, whereas the Salmonella typhimurium (11), Bacillus subtilis (10), and Spinacia oleracea enzymes (15) show Mich...