Characterization of the aspartate kinase from Saccharomyces cerevisiae and of its interaction with threonine.
Aspartate kinase (AK) from Saccharomyces cerevisiae has been characterized to elucidate its quaternary structure and the effect of the allosteric inhibitor threonine on the enzyme conformation. The homogeneously purified enzyme was inhibited by threonine (K(i) 1.4 mM) and was found to bind this compound (K(d) 0.97 mM) in a hyperbolic manner. Gel filtration and native gel electrophoresis indicated that yeast AK is a homohexamer of 346 kDa composed by 58 kDa subunits. Threonine caused a decrease in the apparent molecular mass of AK as evidenced by size-exclusion chromatography (from 345 to 280 kDa) and blue native gel electrophoresis (from 346 to 297 kDa); no other molecular species were detected. This shift in the hydrodynamic size was threonine-specific and was reversed by rechromatography in the absence of threonine. No change in the apparent molecular mass was induced by threonine in an AK mutant insensitive to inhibition by this amino acid, which was observed to be unable to bind threonine. These results indicate that the allosteric transition elicited by binding of threonine to yeast AK involves a large conformational change of the protein that isomerizes from a relaxed active conformation to a more compact inactive one of smaller molecular dimensions.
Marina
Pablo
P
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080 Sevilla, Spain.
Martínez-Costa
Oscar H
OH
Calderón
Isabel L
IL
Aragón
Juan J
JJ
D001222:Aspartate Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D001222:Aspartate Kinase