Cloning, expression, purification, and properties of a putative plasma membrane hexokinase from Solanum chacoense.
A full-length hexokinase cDNA was cloned from Solanum chacoense, a wild relative of the cultivated potato. Analysis of the predicted primary sequence suggested that the protein product, ScHK2, may be targeted to the secretory pathway and inserted in the plant plasma membrane, facing the cytosol. ScHK2 was expressed as a hexahistidine-tagged protein in Escherichia coli. Expression conditions for this construct were optimized using a specific anti-hexokinase polyclonal anti-serum raised against a truncated version of ScHK2. The full-length recombinant protein was purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography followed by anion exchange chromatography on Fractogel EMD DEAE-650 (S). The purified enzyme had a specific activity of 5.3 micromol/min/mg protein. Its apparent Kms for glucose (23 microM), mannose (30 microM), fructose (5.2 mM), and ATP (61 microM) were in good agreement with values found in the literature for other plant hexokinases. Hexahistidine-tagged ScHK2 was highly sensitive to pH variations between 7.7 and 8.7. It was inhibited by ADP and insensitive to glucose-6-phosphate. These findings constitute the first kinetic characterization of a homogeneous plant hexokinase preparation. The relevance of ScHK2 kinetic properties is discussed in relation to the regulation of hexose metabolism in plants.
Claeyssen
Eric
E
Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Rue Sherbrooke est, Montréal, Que., Canada H1X 2B2.
Wally
Owen
O
Matton
Daniel P
DP
Morse
David
D
Rivoal
Jean
J
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006593:Hexokinase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006601:Hexoses / Q000378:metabolism; D008969:Molecular Sequence Data; D032322:Solanum / Q000201:enzymology / Q000235:genetics*
D006601:Hexoses; D006593:Hexokinase