Abstract
The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake. RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K. lactis is required for the up-regulation of glucose transport and also for glucose repression. To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized. Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e. in the presence of substrates and products. The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation. In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration. Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156. The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site. KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity. The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K. lactis.
Highlights
The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake
Signal transduction induced by high external glucose results in a predominance of ScHxk2, whereas expression of ScHXK1 and ScGLK1 is high on non-fermentable carbon sources or galactose [15,16,17]
In contrast to the Crabtree-positive yeast S. cerevisiae [29], in which the glycolytic flux is primarily correlated with the external glucose concentration, regulation of glucose uptake in K. lactis is apparently coupled to oxygen supply [26, 30, 31]
Summary
ScHxk, hexokinase isoenzyme 1 of S. cerevisiae; ScHxk, hexokinase isoenzyme 2 of S. cerevisiae; ScGlk, glucokinase of S. cerevisiae; KlHxk or Rag5p, K. lactis hexokinase; RAG, resistance against antimycin A in the presence of glucose [39]; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; TEA, triethanolamine. K. lactis hexokinase (KlHxk, Rag5p) turned out to be essential for glucose-induced transcription of the low affinity glucose transporter gene RAG1 and the KlPDCA gene encoding pyruvate decarboxylase [35, 38] and was involved in the expression of the high affinity glucose permease Hgt1 [37]. Data are discussed with special consideration of the molecular mechanisms controlling glucose kinase activity of KlHxk including oligomer formation and dissociation and its significance to enzyme function and glucose signaling
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