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Abstract

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.

Data-driven model of glycolysis shows that allostery maintains high ATP levels while mass action controls flux and energy of ATP hydrolysis

“…We calculated the 𝑉 𝑚𝑎𝑥 𝐴/𝐼,𝑟𝑒𝑣 from the other kinetic constants using the Haldane relationship as the 𝑉 𝑚𝑎𝑥 in the reverse direction is rarely measured in the literature. Altogether, structural and regulatory information about HK1 translates into the following general MWC equation for HK1 (note that the simplified kinetic Equation ( 40) was used in the model as described below): Data used for HK1: We manually digitized ~500 data points from eight publications (Bianchi et al, 1998;Copley and Fromm, 1967;Ellison et al, 1975Ellison et al, , 1974Kosow et al, 1973;Ning et al, 1969;Solheim and Fromm, 1983;Ureta et al, 1985) describing the rate of purified HK1 enzyme in the presence of different concentrations of Glucose, ATP, G6P, ADP, and inorganic phosphate (P i ).…”

Single Residue Determinants in the Binding of Recombinant Human Brain Hexokinase to the Mitochondrion

“…Mutations could cause global conformational change in the protein; however, kinetic parameters clearly indicate active enzyme with wild-type properties. Moreover, the removal of residues from the N-terminus has no effect on the kinetic properties of HKI (or hexokinase Type-II) from human and other sources (63,64). Mutations could also change the state of aggregation of HKI, and thereby impede association with the mitochondrion, but constructs here are monomers up to peak concentrations used in mitochondrial binding assays.…”