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-sit e variants of Saccharomyces cerevisiae inorganic pyrophosphatase (PPas e), including measurements of thermostability. oligomeric structure an d 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, D11 7E, D120E and D152E variants had no activity under the conditions test ed, 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 o f Escherichia coli PPase, proving conclusively that the active site an d mechanism of soluble PPases are conserved. The D71E variant was more thermostable and the K56R, R78K, D115E and K154R variants were mon th ermolabile than the wild-type enzyme, whereas subunit:subunit interact ions were somewhat weakened by the K56R, R78K, Y89F and K154R substitu tions. These results suggest that Lys56, Asp71, Arg78, Tyr89, Asp115 a nd Lys154 are structurally important for yeast PPase.