THE CONTRIBUTION OF WATER TO THE SELECTIVITY OF PYRUVATE-KINASE FOR NA+ AND K+

For many years it has been known that K+ is an essential activator of pyruvate kinase [Kachmar, J. F. & Boyer, P. D. (1953) J. Biol. Chem. 2 00, 669-683] and that Na+ induces relatively small enhancements of act ivity. The effect of these two alkali metal ions on the activity of py ruvate kinase entrapped in the low water environment of reverse micell es formed with cetyltrimethylammonium, hexanol, a-octane and various w ater concentrations was studied. In reverse micelles with 3.6% water, the activity with 7 mM Na+ is more than 82 times higher than in aqueou s solution with an equivalent Na+ concentration. As the concentration of water in reverse micelles is raised, the activating effect of relat ively low concentrations of Na+ (or K+) decreases simultaneously to a more than 100-fold increase in the concentration of Na+ or K+ required for attaining half-maximal activation. Similar results were obtained with NH4+, Rb+ and Cs+. Therefore, the amount of water in the system i s critical for observing activation by alkali metal ions. In fact, the concentration of Na+ required for half-maximal activation in standard aqueous media is higher than the concentrations that can be experimen tally assayed. As evidenced from fluorescence and kinetic data, it app ears that the entrapment of pyruvate kinase in reverse micelles does n ot produce gross structural alterations. Therefore, it is suggested th at in conventional aqueous systems, the basis of the high discriminati on between Na+ and K+ by pyruvate kinase is the higher energy required for desolvating Na+. Nevertheless, at all the water concentrations st udied, the activities reached with K+ were higher than with Na+ which suggests that the Na+ form of the enzyme has a lower catalytic capacit y than the K+-enzyme complex.