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.