Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity

J Biol Chem. 2005 Dec 16;280(50):41178-83. doi: 10.1074/jbc.M509324200. Epub 2005 Oct 10.

Abstract

The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Alanine / chemistry
  • Allosteric Site
  • Arabidopsis / enzymology*
  • Aspartate Kinase / chemistry*
  • Aspartic Acid / chemistry
  • Chloroplasts / metabolism
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / metabolism
  • Gene Expression Regulation, Plant*
  • Genes, Reporter
  • Genome, Plant
  • Homoserine Dehydrogenase / chemistry*
  • Isoleucine / chemistry
  • Kinetics
  • Methionine / chemistry
  • Models, Biological
  • Plasmids / metabolism
  • Protein Isoforms
  • Recombinant Proteins / chemistry
  • Spectrophotometry
  • Temperature
  • Threonine / chemistry

Substances

  • DNA, Complementary
  • Enzyme Inhibitors
  • Protein Isoforms
  • Recombinant Proteins
  • Isoleucine
  • Threonine
  • Aspartic Acid
  • Adenosine Triphosphate
  • Methionine
  • Homoserine Dehydrogenase
  • Aspartate Kinase
  • Alanine