Kinetic analysis of yeast galactokinase: implications for transcriptional activation of the GAL genes.
Galactokinase (EC 2.7.1.6) catalyses the first step in the catabolism of galactose. Yeast galactokinase, Gal1p, and the closely related but catalytically inactive Gal3p, also function as ligand sensors in the GAL genetic switch. In the presence of galactose and ATP (the substrates of the reaction catalysed by Gal1p) Gal1p or Gal3p can bind to Gal80p, a transcriptional repressor. This relieves the inhibition of a transcriptional activator, Gal4p, and permits expression of the GAL genes. In order to learn more about the mechanism of ligand sensing by Gal3p and Gal1p, we studied the kinetics of the reaction catalysed by Gal1p. Galactose-1-phosphate, a product of the reaction, is a mixed inhibitor both with respect to galactose and to ATP suggesting that the reaction proceeds via a compulsory, ordered, ternary complex mechanism. There is little variation in either the turnover number or the specificity constants in the pH range 6.0-9.5, implying that no catalytic base is required in the reaction. These data are discussed both in the context of galactokinase enzymology and their implications for the mechanism of transcriptional induction.
Timson
David J
DJ
School of Biological Sciences, The University of Manchester, 2.205 Stopford Building, Oxford Road, M13 9PT, Manchester, UK.
Reece
Richard J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005686:Galactokinase / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005695:Galactosephosphates / Q000096:biosynthesis / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*
D004791:Enzyme Inhibitors; C099289:Gal3 protein, S cerevisiae; D005695:Galactosephosphates; D007554:Isotopes; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D005686:Galactokinase; D005690:Galactose