EQUILIBRIUM MEASUREMENTS OF INTERACTION OF YEAST ENOLASE WITH ACTIVATING METAL IONS

A study has been made of the interaction of yeast enolase (phosphoenolpyruvate hydratase EC 4.2.1.11) with two activating cations, Mn2+ and Mg2+, and one inhibitory cation, Ca2+. In the absence of substrate 2 moles of Mg and Mn are bound per 67,000 molecular weight unit, the molecular weight unit usually present and considered the native molecule. Dissociation constants of 1 × 10-5 and 5 × 104 were found for Mg2+ and 1.3 × 10-6 and 1.9 × 10-5 for Mn2+. Competition studies showed that the tighter bound magnesium ion competes for the same site as the weaker bound manganese ion, although the first site filled by either ion produces the spectral shift reported earlier. In the presence of substrate, at least 2 more moles of either metal ion are bound with considerably weaker affinities. The data show that both substrate and these additional cations must bind to the enzyme alone, but that both substrate and the extra metal ions are bound more tightly in the tertiary complex. The binding of the first 2 moles of cation does not appear to be influenced by the presence of substrate. Calcium ion is found to bind to a multiplicity of sites. Over a threefold range of protein concentration, there was no concentration effect upon the metal dissociation constants. © 1969, American Chemical Society. All rights reserved.