The overexpressed hexahistidine-tagged human hexokinase type III is inhibited by D-glucose.
Inhibition by its product, glucose, is a kinetic property of hexokinase type III. In this paper, we report the overexpression in Escherichia coli of human hexokinase type III. The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end. This modification confers to the product the ability to bind the Ni2+ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups. The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent. Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg. Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose. The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.
Palma
Francesco
F
Istituto di Chimica Biologica "Giorgio Fornaini", Università degli Studi di Urbino, Via A. Saffi, 2, 61029 Urbino (PU), Italy.
Agostini
Deborah
D
Polidori
Emanuela
E
Stocchi
Vilberto
V
D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism*; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D006639:Histidine; D006593:Hexokinase; D005947:Glucose