Hexokinase 2 fromSaccharomyces cerevisiae:  Regulation of Oligomeric Structure byin VivoPhosphorylation at Serine-14

Abstract

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.

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

“…The physiological signal that initiates the covalent modification of residue Ser-15 by a hitherto unidentified protein kinase in S. cerevisiae is glucose limitation (19). Interestingly, it was found only recently that KlHxk1 may be phosphorylated in vivo at the equivalent position Ser-15 and that this modification efficiently stimulates the dissociation of the homodimeric enzyme in vitro, 5 as observed similarly for ScHxk2 (20).…”

“…7B), and thereby promote monomer formation. Assuming that a similar structural situation exists in the homologous hexokinase ScHxk2, the extensive monomer formation observed as a consequence of Ser-15 in vivo phosphorylation or in vitro exchange by glutamate (20) is likely due to the introduction of a negative charge in the intersubunit interface of the dimeric enzyme.…”

“…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.…”

“…Consideration of these numbers, together with the fact that (i) residues involved in protein dimerization are usually surface-exposed in the dissociated state and (ii) surface-exposed amino acids generally tend to be less conserved than amino acids located in the interior of a protein or in the active site, suggests the formation of similar dimers of the three enzymes. In the case of ScHxk2, the phosphorylation of a single amino acid in the dimer interface (Ser-15) is likely to represent an efficient mechanism enabling the cell to induce the dissociation of the hexokinase homodimer, even at the high enzyme concentrations expected to exist in situ (20,21), and to facilitate ScHxk2 interaction with the major nuclear export receptor Xpo1 (9). The physiological signal that initiates the covalent modification of residue Ser-15 by a hitherto unidentified protein kinase in S. cerevisiae is glucose limitation (19).…”

“…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.…”