The electrophilic and leaving group phosphates in the catalytic mechanism of yeast pyrophosphatase.

Binding of pyrophosphate or two phosphate molecules to the pyrophosphatase (PPase) active site occurs at two subsites, P1 and P2. Mutations at P2 subsite residues (Y93F and K56R) caused a much greater decrease in phosphate binding affinity of yeast PPase in the presence of Mn(2+) or Co(2+) than mutations at P1 subsite residues (R78K and K193R). Phosphate binding was estimated in these experiments from the inhibition of ATP hydrolysis at a sub-K(m) concentration of ATP. Tight phosphate binding required four Mn(2+) ions/active site. These data identify P2 as the high affinity subsite and P1 as the low affinity subsite, the difference in the affinities being at least 250-fold. The time course of five "isotopomers" of phosphate that have from zero to four (18)O during [(18)O]P(i)-[(16)O]H(2)O oxygen exchange indicated that the phosphate containing added water is released after the leaving group phosphate during pyrophosphate hydrolysis. These findings provide support for the structure-based mechanism in which pyrophosphate hydrolysis involves water attack on the phosphorus atom located at the P2 subsite of PPase.

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

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) 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 the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.

Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase.

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

Fluoride effects along the reaction pathway of pyrophosphatase: evidence for a second enzyme.pyrophosphate intermediate.

The fluoride ion is a potent and specific inhibitor of cytoplasmic pyrophosphatase (PPase). Fluoride action on yeast PPase during PP(i) hydrolysis involves rapid and slow phases, the latter being only slowly reversible [Smirnova, I. N., and Baykov, A. A. (1983) Biokhimiya 48, 1643-1653]. A similar behavior is observed during yeast PPase catalyzed PP(i) synthesis. The amount of enzyme.PP(i) complex formed from solution P(i) exhibits a rapid drop upon addition of fluoride, followed, at pH 7.2, by a slow increase to nearly 100% of the total enzyme. The slow reaction results in enzyme inactivation, which is not immediately reversed by dilution. These data show that fluoride binds to an enzyme.PP(i) intermediate during the slow phase and to an enzyme.P(i) intermediate during the rapid phase of the inhibition. In Escherichia coli PPase, the enzyme.PP(i) intermediate binds F(-) rapidly, explaining the lack of time dependence in the inhibition of this enzyme. The enzyme.PP(i) intermediate formed during PP(i) hydrolysis binds fluoride much faster (yeast PPase) or tighter (E. coli PPase) than the similar complex existing at equilibrium with P(i). It is concluded that PPase catalysis involves two enzyme.PP(i) intermediates, of which only one (immediately following PP(i) addition and predominating at acidic pH) can bind fluoride. Simulation experiments have indicated that interconversion of the enzyme.PP(i) intermediates is a partially rate-limiting step in the direction of hydrolysis and an exclusively rate-limiting step in the direction of synthesis.

Evolutionary aspects of inorganic pyrophosphatase.

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.

Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase.

Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn24, Ala25, and Asp26 at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg2+ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active site. It is concluded that the metal ion binding site found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ greatly stabilizes the enzyme hexamer but has little effect on catalysis.

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 understood phosphoryl-transfer enzymes and is distinctive in having four divalent metal ions at the active site. Here we determine pH profiles for wild-type Saccharomyces cerevisiae PPase (Y-PPase) and for 14 of its active site variants and consider the effects of active site mutation on the pH-independent parameters and acid dissociation constants that characterize these profiles against the framework of the proposed structure of the activated complex. The results obtained (a) support the current mechanistic model in which a hydroxide ion, stabilized by binding to two metal ions at the active site and by an extended system of hydrogen bonds within the active site, is the nucleophile that attacks enzyme-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 pKa of this group that is a consequence of almost all of the mutations. We further compare the present results to those previously observed for the corresponding mutations in Escherichia coli PPase [E-PPase; Salminen et al. (1995) Biochemistry 34, 782-791]. Such comparison provides a measure of the extent to which different portions of the active site are conserved. We find that some corresponding mutations have different 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, one region of the active site is highly conserved, suggesting that it may represent a common feature of phosphoryl-transfer enzymes or a vestige of a primitive ur-PPase active site.

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.