Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14

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Item Type:	Article
Title:	Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14
Creators Name:	Golbik, R., Naumann, M., Otto, A., Mueller, E.C., Behlke, J., Reuter, R. and Kriegel, T.M.
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 pseudophos-phorylated 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.
Keywords:	Amino Acid Substitution, Dimerization, Enzyme Activation, Enzyme Stability, Glutamic Acid, Hexokinase, Isoenzymes, Site-Directed Mutagenesis, Phosphorylation, Phosphotransferases, Saccharomyces Cerevisiae, Serine, Substrate Specificity
Source:	Biochemistry
ISSN:	0006-2960
Publisher:	American Chemical Society
Volume:	40
Number:	4
Page Range:	1083-1090
Date:	1 January 2001
Official Publication:	https://doi.org/10.1021/bi001745k
PubMed:	View item in PubMed

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