The contribution of water to the selectivity of pyruvate kinase for Na+ and K+.

Abstract

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. 200, 669-683] and that Na+ induces relatively small enhancements of activity. The effect of these two alkali metal ions on the activity of pyruvate kinase entrapped in the low water environment of reverse 'micelles formed with cetyltrimethylammonium, hexanol, n-octane and various water concentrations was studied. In reverse micelles with 3.6% water, the activity with 7 mM Na+ is more than 82 times higher than in aqueous solution with an equivalent Na+ concentration. As the concentration of water in reverse micelles is raised, the activating effect of relatively 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 is 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 experimentally assayed. As evidenced from fluorescence and kinetic data, it appears that the entrapment of pyruvate kinase in reverse micelles does not produce gross structural alterations. Therefore, it is suggested that in conventional aqueous systems, the basis of the high discrimination between Na+ and K+ by pyruvate kinase is the higher energy required for desolvating Na+. Nevertheless, at all the water concentrations studied, the activities reached with K+ were higher than with Na+ which suggests that the Na+ form of the enzyme has a lower catalytic capacity than the K+-enzyme complex.