Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride sugge sts that it replaces active site Mg2+-bound nucleophilic water, for which t wo different locations were proposed previously. To localize the bound fluo ride, we investigate here the effects of mutating Tyr(93) and five dicarbox ylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PPi h ydrolysis and synthesis, formation of enzyme-bound PPi at equilibrium, phos phate-water oxygen exchange, and Mg2+ binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate -limiting in the catalytic cycle, consistent with the mechanism in which th e nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PPi hydrolysis (as characterized by the catalytic constan t and the net rate constant for P-O bond cleavage) were in general larger t han on PPi synthesis (as characterized by the net rate constant for PPi rel ease from active site). The effects of fluoride on the Y-PPase variants con firmed that PPase catalysis involves two enzyme PPi intermediates, which bi nd fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pobjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 119 89-11947). A mechanism for the structural changes underlying the interconve rsion of the enzyme PPi intermediates is proposed.