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PubMed2002

A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi.

The proton pumping pyrophosphatase (H(+)-PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardtii, and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H(+)-PPase in the Golgi complex and plasma membrane of epimastigotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K(+) ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminomethylenediphosphonate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H(+)-PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H(+)-PPase. Taken together, these results are consistent with the presence of a functional H(+)-PPase in the plasma membrane of these parasites.

Animals↗

PubMed1982

Membrane-associated nucleotide pyrophosphatase activity on leucocytes from different compartments.

The distribution of membrane-associated nucleotide pyrophosphatase activity has been investigated on mouse leucocytes of different origin. It is shown that enzyme-specific activity is higher on bone-marrow cells than on cells derived from peripheral organs. Since thymus lymphocytes show the lowest activity, it is obvious that the thymus environment can influence the enzyme activity on bone-marrow stem cells which have entered this organ. Spleen cells from thymus-deficient nude mice displayed more enzyme activity than spleen cells from normal mice, although the percentage of lymphocytes was similar in both donor-cell populations.

Animals↗

PubMed1997

Rat liver ADP-ribose pyrophosphatase-I as an in vitro target of the acetaminophen metabolite N-acetyl-p-benzoquinoneimine.

N-acetyl-p-benzoquinoneimine (NAPQI) is the metabolite responsible for acetaminophen hepatotoxicity. ADP-ribose pyrophosphatase-I (ADPRibase-I; EC 3.6.1.13) hydrolyzes protein-glycating ADP-ribose. The results show NAPQI-dependent alterations of ADPRibase-I leading to strong inhibition: a fast Km increase produced by low concentrations, and a time-dependent Vmax decrease by higher NAPQI concentrations. Both effects were prevented by thiols, but not reverted by them, nor by gel filtration of NAPQI-treated enzyme. Liver ADPRibase-I can be a target of NAPQI-dependent arylation. The inhibition or inactivation of the enzyme would contribute to increasing the free ADP-ribose concentration and nonenzymatic ADP-ribosylation, which is coherent with results linking free ADP-ribose-producing pathways to acetaminophen toxicity.

Acetaminophen↗

PubMed2003

Cloning and expression of the inorganic pyrophosphatase gene from the amino acid producer Brevibacterium lactofermentum ATCC 13869.

A 20-kDa Brevibacterium lactofermentum protein was detected when purifying the His-tagged FtsZBL. The protein was identified by matrix-assisted laser desorption/ionisation time of flight as the inorganic pyrophosphatase encoded by the ppa gene, which is present as a single copy in the genome of Corynebacterium glutamicum. The ppa gene was cloned from B. lactofermentum chromosomal DNA by polymerase chain reaction; it seemed to be an essential gene and it might represent an attractive target for drug discovery. The cloned ppa gene complemented a ppa- Escherichia coli mutant and a ppa-gfp gene fusion revealed that the gene product mainly accumulated at the cell poles in both E. coli and B. lactofermentum.

Base Sequence↗

PubMed1996

Human dUTP pyrophosphatase: uracil recognition by a beta hairpin and active sites formed by three separate subunits.

BACKGROUND: The essential enzyme dUTP pyrophosphatase (dUTPase) is exquisitely specific for dUTP and is critical for the fidelity of DNA replication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis. A high cellular dTTP: dUTP ratio is essential to avoid uracil incorporation into DNA, which would lead to strand breaks and cell death. We report the first detailed atomic-resolution structure of a eukaryotic dUTPase, human dUTPase, and complexes with the uracil-containing deoxyribonucleotides, dUMP, dUDP and dUTP. RESULTS: The crystal structure reveals that each subunit of the dUTPase trimer folds into an eight-stranded jelly-roll beta barrel, with the C-terminal beta strands interchanged among the subunits. The structure is similar to that of the E. coli enzyme, despite low sequence homology between the two enzymes. The nucleotide complexes reveal a simple and elegant way for a beta hairpin to recognize specific nucleic acids: uracil is inserted into a distorted antiparallel beta hairpin and hydrogen bonds entirely to main-chain atoms. This interaction mimics DNA base pairing, selecting uracil over cytosine and sterically precluding thymine and ribose binding. Residues from the second subunit interact with the phosphate groups and a glycine-rich C-terminal tail of the third subunit caps the substrate-bound active site, causing total complementary enclosure of substrate. To our knowledge, this is the first documented instance of all three subunits of a trimeric enzyme supplying residues that are critical to enzyme function and catalysis. CONCLUSIONS: The dUTPase nucleotide-binding sites incorporate some features of other nucleotide-binding proteins and protein kinases, but seem distinct in sequence and architecture. The novel nucleic acid base recognition motif appears ancient; higher order structures, such as the ribosome, may have evolved from a motif of this kind. These uracil-beta-hairpin interactions are an obvious way for peptides to become early coenzymes in an RNA world, providing a plausible link to the protein-DNA world. Within the beta hairpin, there is a tyrosine corner motif that normally specifies beta-arch connections; this tyrosine motif was apparently recruited to discriminate against ribonucleotides, more recently than the evolution of the beta hairpin itself.

Amino Acid Sequence↗

PubMed1997

Some properties of inorganic pyrophosphatase from Bacillus subtilis.

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1; PPase) from Bacillus subtilis was purified to a homogeneous state electrophoretically when analysed by SDS-PAGE. The enzyme consists of six identical subunits; the molecular weight of the native enzyme estimated by gel filtration was approx. 120,000, and denaturing polyacrylamide gel electrophoresis gave a single band corresponding to 24,000. The enzyme absolutely required a divalent cation for its activity. Mg2+ was most effective, showing two steps of concentration-dependent activation. Mg2+ could be partially replaced by Mn2+ and Co2+. The enzyme was thermostable in the presence of Mg2+, and no loss of activity was observed on the incubation at 55 degrees C for an hour.

Bacillus subtilis↗

PubMed1998

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions.

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions, with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following "Michaelian" kinetics with a specific activity of 36.7 U/mg and K0.5 = 88 microM. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited "Michaelian" kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed, K(m) values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K0.5-40 and 90 microM, for magnesium and calcium, respectively), high-affinity sites were also observed with K0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 microM), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed.

Alkaline Phosphatase↗

PubMed1995

Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis?

Using site-directed mutagenesis, we have completed replacing all 17 putative active site residues of Escherichia coli inorganic pyrophosphatase (PPase). We report here the production of 11 new variant proteins and their initial characterization, including thermostability, hydrophobicity, oligomeric structure, and specific activity at pH 8. Studies of the pH-rate profiles of 12 variants containing substitutions for potentially essential residues showed that the effect of the mutation was always to increase the pKa of a basic group essential for both substrate binding and catalysis by 1-3 pH units. The D70E variant had the lowest activity at all pHs; the K29R, R43K, and K142R variants also had low kcat/Km values. The principal effect seen in the other variant proteins was higher and sharper pH optima; their pH-independent kcat and kcat/Km values changed at most by a factor of 8. Our results suggest that the most likely candidate for the essential basic group affected by all mutations in the active site is a hydroxide ion stabilized by coordination to the essential Mg2+ ions. Analyzing our results using the structure recently obtained for E. coli PPase [Kankare et al. (1994) Protein Eng. 7, 823-830] led us to identify a group of residues, centered around Asp70 and including Tyr55, Asp65, Asp67, Asp102, and Lys104, that we believe binds the magnesium ions that are critical for the activity, possibly by stabilizing the essential hydroxide. Others, including Lys29, Arg43, and Lys142, are more spread out and more positively charged. They appear to be involved in binding substrate and product. Tyr55 is also a key part of the hydrophobic core of E. coli PPase; when it or residues that interact with it are conservatively mutated, there are changes in the overall structure of the enzyme as assayed by thermostability, hydrophobicity, or oligomeric structure.

Bacterial Proteins↗

PubMed1995

Effect of D97E substitution on the kinetic and thermodynamic properties of Escherichia coli inorganic pyrophosphatase.

Aspartic acid 97 in the inorganic pyrophosphatase of Escherichia coli (E-PPase) has been identified as an evolutionarily conserved residue forming part of the active site [Cooperman et al. (1992) Trends Biochem. Sci. 17, 262-266]. Here we determine the effect of D97E substitution on several kinetic and thermodynamic properties of E-PPase, including rate and equilibrium constants for enzyme-catalyzed PPi.Pi equilibration at pH 7.2 and 8.0, Mg2+ affinity in the presence and absence of substrate, and the Mg2+ and pH dependence of kcat and Km. We find the major effects of D97E substitution are to (a) decrease markedly the pH-independence rates of both PPi hydrolysis and, especially, PPi resynthesis on the enzyme, (b) selectively destabilize both the EMg4PPi complex and the transition state between this complex and the EMg2(MgPi)2 complex, (c) raise the pKa of the basic group "essential" for PPi hydrolysis and for productive PPi binding by 1.5 and > 2.2 log units, respectively, (d) distort a site to which Mg2+ binds in the absence of substrate such that occupancy of the site by Mg2+ no longer confers enzymatic activity, and (e) decrease the affinity of one of the two Mg2+ ions that binds to enzyme in the presence of substrate. That this multiplicity of effects arises from a single Asp to Glu substitution suggests, in the absence of any evidence for a generalized structural change, a tightly integrated active site in which the perturbation induced by conservative substitution at a single location can have widespread functional effects.

Bacterial Proteins↗

PubMed1984

Isoleucyl-tRNA synthetase from bakers' yeast: variable discrimination between tRNAIle and tRNAVal and different pathways of cognate and noncognate aminoacylation under standard conditions, in the presence of pyrophosphatase, elongation factor Tu-GTP complex, and spermine.

Error rates in discrimination between cognate tRNAIle and noncognate tRNAVal in the aminoacylation reaction with isoleucine catalyzed by isoleucyl-tRNA synthetase from yeast have been investigated in three sets of experiments under different assay conditions. The overall discrimination factor was first determined by isoleucylation of tRNAVal/tRNAIle mixtures. In the second set of experiments, the number of AMP molecules formed per Ile-tRNA in the cognate and noncognate reactions was measured. The higher AMP formation in the noncognate aminoacylation is assigned to a proofreading reaction step. The calculated proofreading factors and an estimated initial discrimination factor yield overall discriminations that are consistent with those obtained from the first set of experiments. In the third series of studies, the orders of substrate addition and product release of cognate and noncognate isoleucylation reactions were investigated by initial rate kinetic methods. From kcat and Km values, the overall discrimination factors were calculated and showed again a good coincidence with those observed in the preceding sets of experiments. Besides under standard assay conditions, aminoacylation reactions were studied in the presence of pyrophosphatase or elongation factor Tu-GTP complex, under addition of both these proteins, in presence of these two additional proteins and spermine at high and low magnesium concentrations, and under special conditions that favor misacylations. Furthermore, isoleucylation of tRNAIle was tested at increased and decreased pH in the standard enzyme assay. Variation of the assay conditions results in changing discrimination factors, which differ by a factor of about 10. Substitution of tRNAIle by tRNAVal in the isoleucylation reaction causes changes in substrate addition and product release orders and thus of the whole catalytic cycle. For aminoacylation of tRNAIle, four different orders of substrate addition and product release appear: the sequential ordered ter-ter, the rapid equilibrium sequential random ter-ter, the random bi-uni uni-bi ping-pong, and a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment. tRNAVal is aminoacylated in rapid equilibrium random ter-ter order, in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment, and in two bi-uni uni-bi ping-pong mechanisms. It is assumed that the different assay conditions can be regarded as a stepwise approximation to physiological conditions and that considerable changes in error rates may be also possible in vivo up to 1 order of magnitude.

Adenosine Monophosphate↗

PubMed1980

Identification of an arginine important for enzymatic activity within the covalent structure of yeast inorganic pyrophosphatase.

Previously we presented evidence for an essential arginine involved in binding inorganic pyrophosphate during catalysis by yeast inorganic pyrophosphatase [Cooperman, B. S., & Chiu, N. Y. (1973b) Biochemistry 12, 1676]. In the present work we show this residue to be arginine-77. Arginine-77 reacts with [14C]phenylgloxal considerably faster than the other five phenylglyoxal is selectively blocked in the presence of the competitive inhibitor calcium pyrophosphate. Our procedure leading to the identification of Arg-77 utilizes the following steps: CNBr cleavage, digestion with Staphylococcus aureus V8 protease and with pepsin, and peptide mapping. All of these steps are performed below pH 5, a restriction imposed by the lability of the phenylglyoxal-arginine adduct at neutral pH. In related work, we find the model compound N alpha-acetyl(diphenylglyoxal)arginine to hydrolyze 10 times more slowly at pH 4 than at pH 7. The high yields of derivatized peptides obtained in this work suggest the potential general utility of our procedure for locating arginine residues derivatized with phenylglyoxal within the covalent structure of proteins.

Amino Acid Sequence↗

PubMed1979

Aminoacyl-tRNA synthetase stimulatory factors and inorganic pyrophosphatase.

A protein was purified from rat liver which stimulated a number of liver aminoacyl-tRNA synthetases. This stimulatory factor was identical with the "tRNA activator" of Dickman & Boll [(1976) Biochemistry 15, 3925] in its mechanism of action and chemical properties, although it was considerably more purified. The two preparations stimulated synthetases by virtue of their pyrophosphatase activity which destroyed the potent inhibitor, PPi, that was present in the reaction mixtures. This PPi was either generated during the reaction or was introduced by contamination of the tRNA or ATP preparations. The degree of inhibition of PPi was strongly influenced by assay conditions, being most effective at low amino acid concentrations, at low pH, and in the presence of heterologous tRNAs. By use of certain assay conditions, PPi concentrations as low as 2 microM could inhibit some synthetases close to 50%. The pitfalls associated with some assay conditions commonly used for aminoacyl-tRNA synthetases are discussed. These studies raise questions about the physiological significance of many previously described aminoacyl-tRNA synthetase stimulatory factors.

Amino Acyl-tRNA Synthetases↗

PubMed2001

Ligand binding sites in Escherichia coli inorganic pyrophosphatase: effects of active site mutations.

Type I soluble inorganic pyrophosphatases (PPases) are well characterized both structurally and mechanistically. Earlier we measured the effects of active site substitutions on pH--rate profiles for the type I PPases from both Escherichia coli (E-PPase) and Saccharomyces cerevisae (Y-PPase). Here we extend these studies by measuring the effects of such substitutions on the more discrete steps of ligand binding to E-PPase, including (a) Mg(2+) and Mn(2+) binding in the absence of added ligand; (b) Mg(2+) binding in the presence of either P(i) or hydroxymethylbisphosphonate (HMBP), a competitive inhibitor of E-PPase; and (c) P(i) binding in the presence of Mn(2+). The active site of a type I PPase has well-defined subsites for the binding of four divalent metal ions (M1--M4) and two phosphates (P1, P2). Our results, considered in light of pertinent results from crystallographic studies on both E-PPase and Y-PPase and parallel functional studies on Y-PPase, allow us to conclude the following: (a) residues E20, D65, D70, and K142 play key roles in the functional organization of the active site; (b) the major structural differences between the product and substrate complexes of E-PPase are concentrated in the lower half of the active site; (c) the M1 subsite is functionally isolated from the rest of the active site; and (d) the M4 subsite is an especially unconstrained part of the active site.

Amino Acid Substitution↗

PubMed2002

Mechanism of the Escherichia coli ADP-ribose pyrophosphatase, a Nudix hydrolase.

Escherichia coli ADP-ribose (ADPR) pyrophosphatase (ADPRase), a Nudix enzyme, catalyzes the Mg(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose 5-phosphate. ADPR hydrolysis experiments conducted in the presence of H(2)(18)O and analyzed by electrospray mass spectrometry showed that the ADPRase-catalyzed reaction takes place through nucleophilic attack at the adenosyl phosphate. The structure of ADPRase in complex with Mg(2+) and a nonhydrolyzable ADPR analogue, alpha,beta-methylene ADP-ribose, reveals an active site water molecule poised for nucleophilic attack on the adenosyl phosphate. This water molecule is activated by two magnesium ions, and its oxygen contacts the target phosphorus (P-O distance of 3.0 A) and forms an angle of 177 degrees with the scissile bond, suggesting an associative mechanism. A third Mg(2+) ion bridges the two phosphates and could stabilize the negative charge of the leaving group, ribose 5-phosphate. The structure of the ternary complex also shows that loop L9 moves fully 10 A from its position in the free enzyme, forming a tighter turn and bringing Glu 162 to its catalytic position. These observations indicate that as part of the catalytic mechanism, the ADPRase cycles between an open (free enzyme) and a closed (substrate-metal complex) conformation. This cycling may be important in preventing nonspecific hydrolysis of other nucleotides.

Amino Acid Sequence↗

PubMed2004

Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii.

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 Sites↗

PubMed2005

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis.

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.

Amino Acid Substitution↗

PubMed2004

Site-specific effects of zinc on the activity of family II pyrophosphatase.

Family II pyrophosphatases (PPases), recently found in bacteria and archaebacteria, are Mn(2+)-containing metalloenzymes with two metal-binding subsites (M1 and M2) in the active site. These PPases can use a number of other divalent metal ions as the cofactor but are inactive with Zn(2+), which is known to be a good cofactor for family I PPases. We report here that the Mg(2+)-bound form of the family II PPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn(2+), but the activity thereafter decays on a time scale of minutes. The activation of the Mn(2+)-form by Zn(2+) was slower but persisted for hours, whereas activation was not observed with the Ca(2+)- and apo-forms. The bound Zn(2+) could be removed from PPase by prolonged EDTA treatment, with a complete recovery of activity. On the basis of the effect of Zn(2+) on PPase dimerization, the Zn(2+) binding constant appeared to be as low as 10(-12) M for S. gordonii PPase. Similar effects of Zn(2+) and EDTA were observed with the Mg(2+)- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn(2+) on the apo- and Mg(2+)-forms of HQ97 and DE15 B. subtilis PPase variants (modified M2 subsite) but not of HQ9 variant (modified M1 subsite) were similar to that for the Mn(2+)-form of wild-type PPase. These findings can be explained by assuming that (a) the PPase tightly binds Mg(2+) and Mn(2+) at the M2 subsite; (b) the activation of the corresponding holoenzymes by Zn(2+) results from its binding to the M1 subsite; and (c) the subsequent inactivation of Mg(2+)-PPase results from Zn(2+) migration to the M2 subsite. The inability of Zn(2+) to activate apo-PPase suggests that Zn(2+) binds more tightly to M2 than to M1, allowing direct binding to M2. Zn(2+) is thus an efficient cofactor at subsite M1 but not at subsite M2.

Bacillus subtilis↗

PubMed2004

Structural studies of metal ions in family II pyrophosphatases: the requirement for a Janus ion.

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.

Bacillus subtilis↗