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 influence of the N-terminal region of human hexokinase type I (HK) on its regulatory 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 for glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P) competitively vs. MgATP with similar Kis (28.5-37 microM). Glucose 1,6-bisphosphate (Glc 1,6-P2) was also a strong inhibitor of all HKs without significant differences among the different truncate forms of the enzyme (Kis 49.5-59 microM). At low concentrations (0-3 mM), Pi was able to reverse the sugar phosphate inhibition of the full-length HK and HK-11aa but not of the 'mini'-HK. In contrast, at high concentrations Pi was an inhibitor of all the hexokinases investigated. These findings confirm that Pi has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-phosphate inhibition by binding to or requiring the N-terminal half of the enzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.
Bianchi
M
M
Institute of Biological Chemistry G Fornaini, University of Urbino, Italy.
Serafini
G
G
Bartolucci
E
E
Giammarini
C
C
Magnani
M
M
D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019298:Glucose-6-Phosphate / Q000037:antagonists & inhibitors; D006593:Hexokinase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D010710:Phosphates / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D013053:Spectrophotometry
D010710:Phosphates; D019298:Glucose-6-Phosphate; D006593:Hexokinase