Magnesium ion requirements for yeast enolase activity

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Abstract
Probably 2 divalent cations are required for enolase [bakers'' yeast] activity, even though the enzyme is a homodimer that specifically binds 4 metal ions in the presence of substrate. A reinvestigation of the stoichiometry of enolase activation is reported. 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. Apparently, the enzyme is inactive when only 2 metal-binding sites are filled and 4 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 Km 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.