A site-directed mutagenesis study of Saccharomyces cerevisiae pyrophosphatase. Functional conservation of the active site of soluble inorganic pyrophosphatases.

We report the expression and initial characterization of 19 active-site variants of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase), including measurements of thermostability, oligomeric structure and specific activity at pH 7.2. 13 of the 19 conservative substitutions resulted in at least a fivefold decrease in activity, indicating that these residues are important for yeast PPase catalysis. The E58D, D117E, D120E and D152E variants had no activity under the conditions tested, suggesting that Glu58, Asp117, Asp120 and Asp152 may have crucial roles in catalysis. The effects of the mutations on catalytic activity were very similar to those observed with the corresponding variants of Escherichia coli PPase, proving conclusively that the active site and mechanism of soluble PPases are conserved. The D71E variant was more thermostable and the K56R, R78K, D115E and K154R variants were more thermolabile than the wild-type enzyme, whereas subunit:subunit interactions were somewhat weakened by the K56R, R78K, Y89F and K154R substitutions. These results suggest that Lys56, Asp71, Arg78, Tyr89, Asp115 and Lys154 are structurally important for yeast PPase.