Alteration of enzyme function of the type II hexokinase C-terminal half on replacements of restricted regions by corresponding regions of glucokinase.
To know the structural properties responsible for the enzymic activity of the 50-kDa C-terminal half of type II hexokinase (HKII-C) derived from rat hepatoma cell line AH130, we constructed cDNAs of HKII-C and its recombinants in which restricted regions containing highly conserved sequences, referred to as regions 2 and 3, were replaced by the corresponding regions of glucokinase. The binding domains of ATP and glucose were proposed to exist in these regions, respectively. Then, the HKII-C and chimera HKII-Cs were overexpressed in Escherichia coli BL21(DE3)pLysS. They all exhibited hexokinase activity, and their activities were inhibited by glucose-6-phosphate (Glc-6-P) competitively for ATP and uncompetitively for glucose. The replacement of region 2 of HKII-C by the corresponding region of glucokinase increased the affinity for glucose and decreased the affinity for Glc-6-P, but it did not significantly affect the affinity for ATP. In contrast, the replacement of region 3 did not cause an appreciable change in hexokinase activity. These findings suggest that region 2 is associated with the binding of ATP and Glc-6-P, and that the latter binding site is located close to the ATP binding site. In addition, region 2 was suggested to be directly related with the binding of glucose and other hexoses.
Kogure
K
K
Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi 1, Tokushima 770, Japan.
Yamamoto
K
K
Majima
E
E
Shinohara
Y
Y
Yamashita
K
K
Terada
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology; D006593:Hexokinase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005958:Glucosephosphates; D011993:Recombinant Fusion Proteins; D019298:Glucose-6-Phosphate; D006593:Hexokinase; D005941:Glucokinase; D005947:Glucose