Regulation of the human-erythrocyte hexose-monophosphate shunt under conditions of oxidative stress. A study using NMR spectroscopy, a kinetic isotope effect, a reconstituted system and computer simulation.
The regulation of the hexose monophosphate shunt of human erythrocytes under conditions of oxidative stress has been investigated by monitoring the reduction of oxidised glutathione (GSSG) to reduced glutathione (GSH) in erythrocytes containing high levels of GSSG; 1H NMR and a biochemical assay were used to measure the changes. A reconstituted metabolic system prepared with the purified erythrocyte enzymes was used in conjunction with studies of intact cells and haemolysates to determine the dependence of the rate of GSH production on the activities of hexokinase and glucose-6-phosphate dehydrogenase. Both of these enzymes have previously been claimed to be the rate-limiting step of oxidatively stimulated flux through the hexose monophosphate shunt. The absence of a kinetic isotope effect on the rate of GSH production in these systems, when [1-2H]glucose replaced glucose as the source of reducing equivalents, showed that glucose-6-phosphate dehydrogenase activity was not a strong determinant of the flux. The dependence of the rate of GSH production on the concentration of the hexokinase inhibitors glucose 1,6-bisphosphate and glycerate 2,3-bisphosphate showed that, under conditions of oxidative stress, hexokinase was the principal determinant of flux through the shunt. Glucose 1,6-bisphosphate at the concentration present in vivo appears to be more important in limiting hexokinase activity, and thus the rate of glucose utilisation, than was previously assumed. A detailed computer model of the system was developed based on the reported kinetic parameters of the enzymes involved. A sensitivity analysis of this model predicted that the hexokinase reaction would have a sensitivity coefficient of 0.995 with respect to the maximal rate of GSH production.
D003201:Computers; D004912:Erythrocytes / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D005978:Glutathione / Q000378:metabolism; D006461:Hemolysis; D006593:Hexokinase / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009243:NAD / Q000032:analysis; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway
D009243:NAD; D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase; D005978:Glutathione; D008274:Magnesium