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J. Biol. Chem., Vol. 278, Issue 41, 39280-39286, October 10, 2003

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The Unique Hexokinase of Kluyveromyces lactis

MOLECULAR AND FUNCTIONAL CHARACTERIZATION AND EVALUATION OF A ROLE IN GLUCOSE SIGNALING^*

Dorit Bär , Ralph Golbik , Gerhard Hübner , Hauke Lilie ¶, Eva-Christina Müller ||, Manfred Naumann **, Albrecht Otto ||, Renate Reuter **, Karin D. Breunig  and Thomas M. Kriegel  

From the Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Institut für Physiologische Chemie, Fetscherstr. 74, D-01307 Dresden, Germany, the Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie, Kurt-Mothes Str. 3, D-06120 Halle/Saale, Germany, the ^¶Martin-Luther-Universität Halle-Wittenberg, Institut für Biotechnologie, Kurt-Mothes Str. 3, D-06120 Halle/Saale, Germany, the ^||Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, D-13092 Berlin, Germany, the ^**Universität Leipzig, Medizinische Fakultät, Institut für Biochemie, Liebigstr. 16, D-04103 Leipzig, Germany, and the Martin-Luther-Universität Halle-Wittenberg, Institut für Genetik, Weinbergweg 10, D-06120 Halle/Saale, Germany

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.

Received for publication, June 2, 2003 , and in revised form, July 21, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant Br921/5 (to K. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Institut für Physiologische Chemie, Fetscherstr. 74, D-01307 Dresden, Germany. Tel.: 49-351-458-6447; Fax: 49-351-458-6306; E-mail: kriegel{at}rcs.urz.tu-dresden.de.

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