A recombinant human 'mini'-hexokinase is catalytically active and regulated by hexose 6-phosphates.
Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion. According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule. We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917. This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c. The overexpressed 'mini'-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme. These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.
Magnani
M
M
Istituto di Chimica Biologica Giorgio Fornaini, Università degli Studi di Urbino, Italy.
Bianchi
M
M
Casabianca
A
A
Stocchi
V
V
Daniele
A
A
Altruda
F
F
Ferrone
M
M
Silengo
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006593:Hexokinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006600:Hexosephosphates / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010920:Placenta / Q000187:drug effects / Q000201:enzymology; D011247:Pregnancy; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D006600:Hexosephosphates; D011994:Recombinant Proteins; D004247:DNA; D006593:Hexokinase