Hexokinases are key enzymes in the metabolism of glucose, fructose and mannose. They are also involved in glucose signalling in eukaryotes from yeast to man, including plants (Entian, 1980
; Rolland et al.
; Wilson, 2003
). The budding yeast Saccharomyces cerevisiae
has three ‘genuine’ glucose kinases (ScHxk1, ScHxk2 and ScGlk1) encoded by the genes ScHXK1
, respectively. Expression of any of these kinases alone is sufficient to allow growth on glucose (Lobo & Maitra, 1977
). In contrast, the glucokinase paralogue ScEMI2
nduction; Enyenihi & Saunders, 2003
) encodes a protein which, although exhibiting 72% identity to ScGlk1, is apparently unable to support glucose utilization of a hxk1 hxk2 glk1
triple kinase mutant of S. cerevisiae
(Vojtek & Fraenkel, 1990
). Isoenzyme ScHxk2 regulates the expression of ScHXK1
(Rodriguez et al.
) and plays a prominent role in glucose sensing and glucose repression (Moreno et al.
). Glucose limitation results in phosphorylation of ScHxk2 at Ser14 in vivo
(Kriegel et al.
), causing dissociation of the homodimeric enzyme in vitro
(Behlke et al.
). Monomeric ScHxk2 shows increased substrate affinity and, in contrast to the dimeric enzyme, is inhibited by free adenosine 5′-triphosphate (ATP) at physiological concentrations (Golbik et al.
). ScHxk2 interacts via
residues 6–15 with the repressor protein ScMig1 to form a complex that mediates glucose repression in the nucleus (Moreno et al.
). The significance of phosphorylation of Ser14 and dissociation of the homodimeric enzyme to form ScHxk2–ScMig1, however, has not yet been experimentally addressed.
The existence of three glucose-phosphorylating enzymes (ScHxk1, ScHxk2 and ScGlk1) and one glucokinase-like protein (ScEmi2) in S. cerevisiae
reflects genetic redundancy resulting from a whole genome-duplication event in the evolutionary history of the genus Saccharomyces
(Wolfe & Shields, 1997
; Seoighe & Wolfe, 1999
). In contrast, the genome of Kluyveromyces lactis
which did not undergo duplication encodes a single hexokinase (KlHxk1; Bär et al.
) and a single glucokinase (KlGlk1; Kettner et al.
). Monomeric KlHxk1 has a molecular weight of about 53 kDa and shares 70% and 73% sequence identity and 84% and 85% sequence similarity with ScHxk1 and ScHxk2, respectively. The enzyme predominantly exists as a homodimer at protein concentrations higher than 1 mg ml−1
(Bär et al.
). Unlike ScHxk2, glucose phosphorylation by KlHxk1 is not inhibited by free ATP at cellular levels of the nucleotide (Bär et al.
). Interestingly, autophosphorylation of KlHxk1, presumably affecting serine residue 156, only results in partial inactivation of the enzyme (Bär et al.
), while ScHxk2 is completely inactivated by autophosphorylation of the equivalent residue Ser157, which is located in the immediate vicinity of the active site (Heidrich et al.
). Despite significant progress in the understanding of the catalytic and regulatory functions of hexokinases at the molecular level, no high-resolution dimer structure of any yeast hexokinase has been reported to date. Structure determination of the KlHxk1 homodimer is expected to provide the molecular data required to obtain novel insights into the control of enzyme conformation, catalytic activity and interaction with cytosolic and nuclear proteins of K. lactis
by modulation of the monomer–homodimer equilibrium.