Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine

Abstract

The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-α helix-loop-β strand-loop-β strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wildtype and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln443 and Gln524. Our results also demonstrated that threonine interaction with Gln443 leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln524. Interaction of this second threonine with Gln524 leads to inhibition of homoserine dehydrogenase activity.