ArticleFaller LD, Baroudy BM, Johnson AM, Ewall RX.
Biochemistry. 1977 Aug 23;16(17):3864-9.
It has generally been concluded that two divalent cations are required for enolase activity, even though the enzyme is a homodimer that specifically binds four metal ions in the presence of substrate. This paper reports a reinvestigation of the stoichiometry of enolase activation. Specific ion electrode measurements of Mg2+ binding in the presence and absence of substrate are compared with stopped-flow measurements of the velocity of 2-phosphoglycerate dehydration. It is concluded that the enzyme is inactive when only two metal-binding sites are filled and that four sites must be populated with Mg2+ for full activity. An ordered binding mechanism is proposed that quantitatively predicts the activation of enolase by the four Mg2+ ions from their measured dissociation constants and the Michaelis constant for the dehydration reaction. To explain the loss of enzymatic activity at still higher metal concentrations, the binding of additional, inhibitory Mg2+ ions is postulated.