KINETIC STUDIES ON ACTIVATION OF YEAST ENOLASE BY DIVALENT CATIONS

Activation constants for Mg2+ and Mn2+ have been determined for yeast enolase at several concentrations of 2-phosphoglyceric acid in the forward reaction and phosphoenolpyruvic acid in the reverse direction. Michaelis constants for both substrates have been determined at several concentrations of the activating cations. The activation constants are shown to be independent of substrate concentration and the Michaelis constants are independent of metal ion concentration. It is concluded that activating ions and substrates bind to the critical sites independently, ruling out the possibility that the metal functions as a bridge to bind substrate and enzyme. The kinetically determined activation constants were found to be nearly the same as the binding constants for the two cations at the weak sites, determined by equilibrium dialysis. Inhibition by excess Mn2+ is shown to be dependent upon the concentration of substrate. The inhibition constant is similar to the binding constant for the binding of a third Mn2+ in the presence of substrate. © 1969, American Chemical Society. All rights reserved.