title: Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14.
abstract: Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomyces cerevisiae. The degree of in vivo phosphorylation of hexokinase 2 at serine-14 is inversely related to the extracellular glucose concentration [Vojtek, A. B., and Fraenkel, D. G. (1990) Eur. J. Biochem. 190, 371-375]; however, a physiological role of the modification causing the dissociation of the dimeric enzyme in vitro [as effected by a serine-glutamate exchange at position 14; Behlke et al. (1998) Biochemistry 37, 11989-11995] is unclear. This paper describes a comparative stopped-flow kinetic and sedimentation equilibrium analysis performed with native unphosphorylated hexokinase 2 and a permanently pseudophosphorylated glutamate-14 mutant enzyme to determine the functional consequences of phosphorylation-induced enzyme dissociation. The use of a dye-linked hexokinase assay monitoring proton generation allowed the investigation of the kinetics of glucose phosphorylation over a wide range of enzyme concentrations. The kinetic data indicated that monomeric hexokinase represents the high-affinity form of isoenzyme 2 for both glycolytic substrates. Inhibition of glucose phosphorylation by ATP [Moreno et al. (1986) Eur. J. Biochem. 161, 565-569] was only observed at a low enzyme concentration, whereas no inhibition was detected at the high concentration of hexokinase 2 presumed to occur in the cell. Pseudophosphorylation by glutamate substitution for serine-14 increased substrate affinity at high enzyme concentration and stimulated the autophosphorylation of isoenzyme 2. The possible role of hexokinase 2 in vivo phosphorylation at serine-14 in glucose signaling is discussed.
authors.affiliation: Martin-Luther-Universität Halle-Wittenberg, Fachbereich Biochemie/Biotechnologie, Institut für Biochemie, Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Golbik
authors.forename: M
authors.initials: M
authors.lastname: Naumann
authors.forename: A
authors.initials: A
authors.lastname: Otto
authors.forename: E
authors.initials: E
authors.lastname: Müller
authors.forename: J
authors.initials: J
authors.lastname: Behlke
authors.forename: R
authors.initials: R
authors.lastname: Reuter
authors.forename: G
authors.initials: G
authors.lastname: Hübner
authors.forename: T M
authors.initials: TM
authors.lastname: Kriegel
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D007527:Isoenzymes; D018698:Glutamic Acid; D012694:Serine; D017853:Phosphotransferases (Alcohol Group Acceptor); D006593:Hexokinase