We report the first crystal structures of a family II pyrophosphatase complexed with a substrate analogue, imidodiphosphate (PNP). These provide new insights into the catalytic reaction mechanism of this enzyme family. We were able to capture the substrate complex both by fluoride inhibition and by site-directed mutagenesis providing complementary snapshots of the Michaelis complex. The universally present inorganic pyrophosphatase (PPase, EC 3.6.1.1) 4 is a central enzyme of phosphorus metabolism.PPases are essential enzymes, because they hydrolyze the inorganic pyrophosphate (PP i ) generated during a number of ATPdependent cellular processes and thus provide the necessary thermodynamic pull for them (1). PPases require divalent metal cations for catalysis. Soluble PPases comprise two families, which differ completely in both sequence (2, 3) and structure (4, 5). Family I PPases (reviewed in Ref. 6) occur in all types of cells from bacteria to man, whereas family II PPases occur almost exclusively in bacteria. Of the 57 known family II PPases, 53 occur in eubacteria, 3 in archaebacteria, and 1 in a unicellular eukaryote (Giardia lamblia). Three Vibrio species, including Vibrio cholerae, have genes for both family I and family II PPases. The frequent occurrence of family II PPase in human pathogens (e.g. Streptococcus agalactiae causes neonatal pneumonia, sepsis, and meningitis; Streptococcus mutans, dental caries; and V. cholerae, cholera) makes studies of this enzyme medically important. In addition, family II PPases belong to the "DHH" family of phosphoesterases, named after the characteristic DHH amino acid signature (7). All of these enzymes have similar structures (5, 8) and presumably related catalytic mechanisms.In contrast to family I PPases, which have a simple cup-like single-domain structure, family II PPases have two domains, with the active site at the domain interface (4, 5) (Fig. 1A). The C-terminal domain of family II PPase contains the high affinity substrate-binding site, whereas the catalytic site that binds the nucleophile-coordinating metal cations is located in the N-terminal domain. Closure of the C-terminal domain onto the N-terminal one creates the catalytically competent conformation by bringing the electrophilic phosphate of substrate into the catalytic site (9). The trigger for domain closure is substrate binding to the C-terminal domain in the open conformation (9).The two PPase families catalyze the hydrolysis of PP i in what initially appeared to be similar active sites (4, 5) (Fig. 1B), but their functional properties are significantly different. The natural metal cofactor of family I PPases is Mg 2ϩ , binding to the enzyme with micromolar affinity, whereas family II enzymes are best activated by Mn 2ϩ or Co 2ϩ , which bind with nanomolar affinity. With these metal ions, family II PPases are ϳ10-fold more active than family I PPases (k cat of 1700 -3300 s Ϫ1 versus 110 -330 s Ϫ1 ) (10 -12). Interestingly, Mg 2ϩ confers lower activity but greater substrate-binding affinity on fam...

Family II inorganic pyrophosphatases (PPases) constitute a new evolutionary group of PPases, with a different fold and mechanism than the common family I enzyme; they are related to the "DHH" family of phosphoesterases. Biochemical studies have shown that Mn(2+) and Co(2+) preferentially activate family II PPases; Mg(2+) partially activates; and Zn(2+) can either activate or inhibit (Zyryanov et al., Biochemistry, 43, 14395-14402, accompanying paper in this issue). The three solved family II PPase structures did not explain the differences between the PPase families nor the metal ion differences described above. We therefore solved three new family II PPase structures: Bacillus subtilis PPase (Bs-PPase) dimer core bound to Mn(2+) at 1.3 A resolution, and, at 2.05 A resolution, metal-free Bs-PPase and Streptococcus gordonii (Sg-PPase) containing sulfate and Zn(2+). Comparison of the new and old structures of various family II PPases demonstrates why the family II enzyme prefers Mn(2+) or Co(2+), as an activator rather than Mg(2+). Both M1 and M2 undergo significant changes upon substrate binding, changing from five-coordinate to octahedral geometry. Mn(2+) and Co(2+), which readily adopt different coordination states and geometries, are thus favored. Combining our structures with biochemical data, we identified M2 as the high-affinity metal site. Zn(2+) activates in the M1 site, where octahedral geometry is not essential for catalysis, but inhibits in the M2 site, because it is unable to assume octahedral geometry but remains trigonal bipyramidal. Finally, we propose that Lys205-Gln81-Gln80 form a hydrophilic channel to speed product release from the active site.

Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn(2+) rather than Mg(2+), the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and II PPases. We measured rates of PP(i) hydrolysis, PP(i) synthesis, and P(i)/water oxygen exchange catalyzed by sgPPase with Mn(2+), Mg(2+), or Co(2+) in the high-affinity metal-binding site and Mg(2+) in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P(i)). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PP(i) complex isomerization known to occur in family I PPases; (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PP(i) hydrolysis and product release; (c) the marked acceleration of sgPPase catalysis in the presence of Mn(2+) and Co(2+) results from a combined effect of these ions on bound PP(i) hydrolysis and P(i) release; (d) sgPPase exhibits lower affinity for both PP(i) and P(i); and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K(m), which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PP(i) concentrations).

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.