EQUILIBRIUM AND KINETIC STUDIES ON REVERSIBLE DISSOCIATION OF YEAST ENOLASE BY NEUTRAL SALTS

The dissociation of yeast enolase by KCl has been confirmed by the preparation of hybrids of native and acetylated enzyme and by sedimentation equilibrium and gel filtration studies. Examination of the equilibrium between inactive monomers and native enzyme at different concentrations of protein, salt, and magnesium ion show that the equilibrium we observe is a monomer-dimer equilibrium and that the effect of chloride or bromide ion fits a mass action law over the observable range of the equilibrium. The greater dissociating power of bromide is attributable to a greater number of binding sites for bromide. Acetate is shown to have no measurable dissociating effect while perchlorate and nitrate have effects stronger than bromide or iodide. The effect of magnesium on the equilibrium is not readily fitted to a simple model, although it pulls the equilibrium strongly toward active dimer formation. Substrate promotes association, but again the effects are not easily interpreted. The dissociation reaction was found to be first order in protein, and Arrhenius plots are shown for dissociation at different salt concentrations at different magnesium and substrate concentrations. The data indicate that the rate of dissociation is unaffected by the ionic environment. Association reactions are found to be second order initially, and activation parameters have been calculated. Temperature effects on dissociation rates, but not association rates, show a large change in heat capacity for the activation process, similar to cited protein denaturation reactions and indicating exposure of hydrophobic residues to solvent in the monomeric state. © 1969, American Chemical Society. All rights reserved.