EVOLUTIONARY CONSERVATION OF ENZYMATIC CATALYSIS - QUANTITATIVE COMPARISON OF THE EFFECTS OF MUTATION OF ALIGNED RESIDUES IN SACCHAROMYCES-CEREVISIAE AND ESCHERICHIA-COLI INORGANIC PYROPHOSPHATASES ON ENZYMATIC-ACTIVITY

Soluble inorganic pyrophosphatase (PPase) is one of the better underst ood phosphoryl-transfer enzymes and is distinctive in having four diva lent metal ions at the active site. Here we determine pH profiles for wild-type Saccharomyces cerevisiae PPase (Y-PPase) and for 14 of its a ctive site variants and consider the effects of active site mutation o n the pH-independent parameters and acid dissociation constants that c haracterize these profiles against thr framework of the proposed struc ture of the activated complex. The results obtained (a) support the cu rrent mechanistic model in which a hydroxide ion, stabilized by bindin g to two metal ions at the active site and by an extended system of hy drogen bonds within the active site, is the nucleophile that attacks e nzyme-bound inorganic pyrophosphate and (b) provide evidence that the acid group that is necessary for maximal activity is a water molecule coordinated to a third metal ion, as shown by the general rise in the pK(a) of this group that is a consequence of almost all of the mutatio ns. We further compare the present results to those previously observe d for the corresponding mutations in Escherichia coli PPase [E-PPase; Salminen et al. (1995) Biochemistry 34, 782-791]. Such comparison prov ides a measure of the extent to which different portions of the active site are conserved. We find that some corresponding mutations have di fferent effects on catalytic function, demonstrating that even in the context of very similar active sites, interactions of the mutated site with less well conserved portions of the enzyme, in this case outside the active site, can lead to different outcomes. On the other hand, o ne region of the active site is highly conserved, suggesting that it m ay represent a common feature of phosphoryl-transfer enzymes or a vest ige of a primitive ur-PPase active site.