Hexokinase of Human Erythrocytes

Abstract

Hexokinase was purified 680-fold from erythrocytes of man. The preparation was not contaminated by other glycolytic enzymes.Initial reaction rate measurements were performed at pH 7.2, ionic strength 0.15 and 37 "C by use of coupled optical assays. The experimental data were used to construct a kinetic model of the enzyme, the kinetic parameters of which were estimated by means of a computer-fit program.The hexokinase of erythrocytes operates by a rapid-equilibrium random mechanism. The dissociation constant of the E . glucose complex is 0.038 to 0.054 mM, of the F, -MgATP complex 1.0 to 2.1 mM. Uncomplexed Mg2+ up to 4 mM is capable of activating the enzyme velocity twofold (Knrgl+ = 1.0 mM). An inhibition was observed a t higher Mg2+ concentrations. a-D-Glucose 6-phosphate and &-D-glUCoSe 1,6-bisphosphate inhibit the hexokinase competitive to MgATP with dissociation constants of 0.069 mM. The enzyme is also inhibited by 2,3-bisphosphoglycerate competitive with respect to MgATP with a K P~G = 2.7 mM. An inhibition by uncomplexed ATP was not detected. The parameters of the kinetic model of the enzyme in conjunction with the free intracellular Concentrations of substrates and effectors were used to calculate the actual intracellular activity of the hexokinase in dependence on variations of pH, temperature and the state of haemoglobin oxygenation. The calculated hexokinase activities correspond to the glycolytic flux in all conditions considered, except a t pH 8.

Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement

“…This is in agreement with the finding of Rapoport et al [43] that the concentration of ATP in erythrocytes incubated with glucose (Glc) at different pH values is constant ; this finding was also reported by Jacobasch et al [35]. Note, however that Minakami and Yoshikawa [37] have reported a pH-dependence of ATP concentration.…”

“…At pH 6.9 Minakami and Yoshikawa [37] measured a decrease in glycolytic rate of 57 % relative to pH 7.2 ; a decrease of 55 % was predicted by the model. At pH 7.5, those authors measured an increase in glycolytic rate of 46 %, while the model predicted an increase of 53 %.…”

“…PFK was modelled with a concerted twostate symmetry scheme [6] ; it is a modification of the two- [46] ; c K eq l p/a; value measured at pH 8.0, 38 mC [47] ; d k cat,r was calculated from the specific activity of the purified (51100i) human erythrocyte enzyme (843 units/mg at pH 8.3, 30 mC ; [48]) adjusted to pH 7.2 by fitting eqn. (A2) to data for the pH dependence of GPI activity [95] and adjusted to 37 mC with the temperature correction factor v (30 mC) l 0.76iv(37 mC) [37] ; GPI is reported to be a dimer of identical subunits with a total M r of 132 000p2000 [48] ; e the specific activity of the reverse reaction was found to be 1.2 times that of the forward reaction [45,47] ; f the concentration of GPI in human erythrocytes, e o , was estimated from the measured human erythrocyte activity (20 064 units/litre of erythrocytes in the reverse direction, pH 7.6, 37 mC ; [37,62] [7]. Hence the proportion of PFK in the R state is given by :…”

Regulation of human erythrocyte hexokinase by glucose‐1, 6‐diphosphate and inorganic phosphate

“…It is involved in glycolysis by its positive effects on phosphofructokinase [4] and pyruvate kinase [5] and by its inhibitory action on hexokinase [6,7]. The inhibition constants reported for hexokinase type I from human erythrocytes [6,7] are well in or below the range of the intracellular concentrations [l-3], suggesting a role for Glc-1 ,6-PZ in-regulation of hexokinase activity in vivo. In contrast Beitner et al [8] reported that Glc-1,6-P* exerted a selective inhibitory effect on hexokinase type II and had no effect on type I hexokinase.…”

A Mathematical Model for the Influence of Fructose 6‐Phosphate, ATP, Potassium, Ammonium and Magnesium on the Phosphofructokinase from Rat Erythrocytes

“…Therefore, the inhibition by magnesium ions becomes visible only after the level of free ATP is negligible. With respect to the influence of magnesium ions, phosphofructokinase appears to differ from hexokinase [21] and pyruvate kinase [22] which show an activation by magnesium. However, there are some indications that the effect on pyruvate kinase is also essentially a deinhibition [23] of the enzyme caused by free ATP.…”