Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression.

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.