title: The unique hexokinase of Kluyveromyces lactis. Molecular and functional characterization and evaluation of a role in glucose signaling.
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.
authors.affiliation: Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Institut für Physiologische Chemie, Fetscherstr. 74, D-01307 Dresden, Germany.
authors.forename: Dorit
authors.initials: D
authors.lastname: Bar
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.forename: Gerhard
authors.initials: G
authors.lastname: Hübner
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Eva-Christina
authors.initials: EC
authors.lastname: Müller
authors.forename: Manfred
authors.initials: M
authors.lastname: Naumann
authors.forename: Albrecht
authors.initials: A
authors.lastname: Otto
authors.forename: Renate
authors.initials: R
authors.lastname: Reuter
authors.forename: Karin D
authors.initials: KD
authors.lastname: Breunig
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Kriegel
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D012694:Serine; D006593:Hexokinase; D005947:Glucose