Regulation of Phosphotransferase Activity of Hexokinase 2 fromSaccharomycescerevisiaeby 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.

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

“…In detail, crystal form VIII was grown in the presence of the glucose analog D-xylose and ATP, whereas crystal form IX was grown in the presence of the nonhydrolyzable ATP analog AMP-PCP. In accordance with the dissociative effect exerted by glucose and glucose 6-phosphate on both KlHxk1 and ScHxk2 (21,23), the presence of (pseudo)substrates may increase the partial concentration of the monomer. Third, a proteolytic removal of N-terminal amino acids known to stabilize the dimeric structure of the homologous hexokinase ScHxk2 (61) cannot be excluded as a cause for monomer formation during long-term crystallization.…”

“…In contrast, no data are available on the covalent modification and/or intracellular distribution of KlHxk1 in the nonfermentative yeast K. lactis. The two hexokinases exhibit 73% sequence identity (22), and dimerization of the K. lactis enzyme apparently decreases its glucose kinase activity (23), as observed similarly for ScHxk2 (21). In addition, the monomer-dimer equilibrium of both hexokinases depends in a comparable manner on the enzyme concentration and on the presence of substrates and products with glucose most strongly inducing homodimer dissociation (21,23).…”

“…2) suggest the existence of the ring-shaped dimeric enzyme also under physiological conditions. In particular, the specific subunit assembly of the symmetric KlHxk1 dimer provides a structural basis that allows to understand on a molecular level the phenomena that were observed for both S. cerevisiae and K. lactis hexokinases: the dependence of glucose kinase activity on the enzyme concentration and oligomeric state, respectively (21,23), the influence of substrates and other ligands on the monomer-dimer equilibrium (21,23), and the impact of Ser-15 phosphorylation on hexokinase dimerization (20) and putative regulatory functions (9,21,23). These phenomena will be discussed in detail in the following paragraphs considering structure-function interrelations.…”

“…The glucose kinase activities of KlHxk1 and ScHxk2 have been shown to depend on the enzyme concentration, with the dimeric species being apparently less active (21,23). It is currently unclear whether the dimer is completely inactive and catalytic activity is exclusively associated with the fraction of monomeric enzyme being in equilibrium with the dimer or whether the dimeric hexokinase displays a reduced activity compared with the monomer.…”

“…Because of the observation that glucose limitation stimulates both the phosphorylation of ScHxk2 at Ser-15 in vivo (18,19) (which in vitro causes the dissociation of the homodimeric enzyme (20)) and its export from the nucleus (9), the covalent modification at Ser-15 and subsequent changes of the oligomeric state have been presumed to be involved in the control of ScHxk2 regulatory functions (9,21). In contrast, no data are available on the covalent modification and/or intracellular distribution of KlHxk1 in the nonfermentative yeast K. lactis.…”