Activation of yeast enolase by Cd(II)

Abstract

Activation of yeast enolase by Cd 2+ exhibits properties similar to activation by the physiological cofactor Mg 2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd 2+ concentration. The effect of pH on activity produced by Cd 2+ is very similar to that produced by Mg 2+, except that the Cd 2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd 2+-enzyme is about 10% of that of the Mg 2+-enzyme. The apparent kinetic parameters of Cd 2+ activation show little effect of pH except for inhibition by high concentrations of Cd 2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd 2+ being a less effective “catalytic” metal ion, and Cd 2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd 2+ - enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg 2+ or Cd 2+ as cofactor. The slowness of the Cd 2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd 2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd 2+ to the catalytic sites is characterized by low association and dissociation rate constants.