Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine.
The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta 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 wild-type 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 Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.
Paris
Stéphane
S
Unité Mixte de Recherche 1932, Laboratoire Mixte CNRS/Institut National de la Recherche Agronomique/Bayer Cropscience, 14-20 Rue Pierre Baizet, 69263 Lyon, France.
Viemon
Claire
C
Curien
Gilles
G
Dumas
Renaud
R
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001225:Aspartokinase Homoserine Dehydrogenase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013912:Threonine
D010940:Plant Proteins; D013912:Threonine; D001225:Aspartokinase Homoserine Dehydrogenase