Enzymatic properties of overexpressed human hexokinase fragments

Full-length hexokinase (HK; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) , a truncate form of the enzyme lacking the first 11 amino acids (HK-11aa) and the 50 kDa C-terminal half ('mini'-HK) containing the catalytic domain, were overexpressed and purified to homogeneity to investigate the influenc e of the N-terminal region of human hexokinase type I (HK) on its regulator y properties. All forms of the enzyme are catalytically active with the HK- 11aa being the most active. All the forms of HK showed the same affinity fo r glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P ) competitively vs. MgATP with similar K(i)s (28.5-37 mu M). Glucose 1,6-bi sphosphate (Glc 1,6-P-2) was also a strong inhibitor of all HKs without sig nificant differences among the different truncate forms of the enzyme (K(i) s 49.5-59 mu M). At low concentrations (0-3 mM), P-i was able to reverse th e sugar phosphate inhibition of the full-length HK and HK-11aa but not of t he 'mini'-HK. In contrast, at high concentrations P-i was an inhibitor of a ll the hexokinases investigated. These findings confirm that P-i has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-p hosphate inhibition by binding to or requiring the N-terminal half of the e nzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.