Regulation of Phosphotransferase Activity of Hexokinase 2 from Saccharomyces cerevisiae by Modification at Serine-14
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- 作者：Ralph Golbik ; Manfred Naumann ; Albrecht Otto ; Eva-Christina Mü ; ller ; Joachim Behlke ; Renate Reuter ; Gerhard Hü ; bner ; and Thomas M. Kriegel
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：January 30, 2001
- 年：2001
- 卷：40
- 期：4
- 页码：1083 - 1090
- 全文大小：103K
- 年卷期：v.40,no.4(January 30, 2001)
- ISSN：1520-4995
文摘
Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomycescerevisiae. The degree of in vivo phosphorylation of hexokinase 2 at serine-14 is inversely related to theextracellular 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 enzymein 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 sedimentationequilibrium 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 generationallowed the investigation of the kinetics of glucose phosphorylation over a wide range of enzymeconcentrations. The kinetic data indicated that monomeric hexokinase represents the high-affinity formof isoenzyme 2 for both glycolytic substrates. Inhibition of glucose phosphorylation by ATP [Moreno etal. (1986) Eur. J. Biochem. 161, 565-569] was only observed at a low enzyme concentration, whereasno 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 enzymeconcentration and stimulated the autophosphorylation of isoenzyme 2. The possible role of hexokinase 2in vivo phosphorylation at serine-14 in glucose signaling is discussed.