RESUMO
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.
Assuntos
Arabidopsis/enzimologia , Aspartoquinase Homosserina Desidrogenase/análise , Sequência de Aminoácidos , Arabidopsis/genética , Aspartoquinase Homosserina Desidrogenase/genética , Aspartoquinase Homosserina Desidrogenase/metabolismo , Ativação Enzimática/genética , Cinética , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Relação Estrutura-Atividade , TreoninaRESUMO
A proteolytic fragment (Mr approximately 25 000) carrying only the aspartokinase activity has been purified by chromatofocusing after limited proteolysis of aspartokinase I-homoserine dehydrogenase I from E.coli K12. The NH2-terminal sequence shows that it corresponds to the amino terminal peptide of the native enzyme. The results confirm a previous hypothesis about the organization of native aspartokinase I-homoserine dehydrogenase I.
Assuntos
Aspartato Quinase/isolamento & purificação , Aspartoquinase Homosserina Desidrogenase/análise , Escherichia coli/enzimologia , Complexos Multienzimáticos/análise , Fosfotransferases/isolamento & purificação , Sequência de Aminoácidos , Cromatografia , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Pronase/metabolismoRESUMO
Limited proteolysis of aspartokinase I-homoserine dehydrogenase I from Escherichia coli by type VI protease from Streptomyces griseus yields five proteolytic fragments, three of which are dimeric, the other two being monomeric. One of the monomeric fragments (27 kilodaltons) exhibits residual aspartokinase activity, while the second one (33 kilodaltons) possesses residual homoserine dehydrogenase activity. The smallest of the dimeric species (2 X 25 kilodaltons) is inactive; the two other dimers exhibit either only homoserine dehydrogenase activity (2 X 59 kilodaltons) or both activities (hybrid fragment, 89 + 59 kilodaltons). This characterization of the proteolytic species in terms of molecular weight, subunit structure, and activity leads to the proposal of a triglobular model for the native enzyme. In addition, the time course of the formation of the various fragments was followed by measuring enzymatic activity and performing gel electrophoretic analysis of the protein mixture at defined time intervals during proteolysis. On the basis of the results of these studies, a reaction scheme describing the succession of events during proteolysis is given.
