The promiscuous ectonucleotidase NPP1: molecular insights into substrate binding and hydrolysis

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) represents the main subtype of the NPP family of nucleotide hydrolyzing enzymes. The ecto-enzyme hydrolyzes structurally diverse substrates and has recently been proposed as a drug target for immuno-oncology. To get more insights into the nature of the promiscuity of NPP1, we investigated its substrate preferences employing a broad range of natural nucleotides including ATP, UTP, diadenosine tetraphosphate (AP₄A), cAMP, and cyclic guanosine-(2′,5′)-monophosphate-adenosine-(3″,5″)-monophosphate (2′,3″-cGAMP), as well as the artificial substrate p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP). Despite their diverse structures, all substrates were converted to nucleoside 5′-monophosphates; 2′,3″-cGAMP yielded exclusively the nucleoside 5′-monophosphates AMP and GMP. In contrast, 3′,3″-bridged cyclic dinucleotides were not hydrolyzed. ATP was the most efficiently hydrolyzed substrate of NPP1, followed by AP₄A and 2′,3″-cGAMP. UTP, cAMP and p-Nph-5′-TMP were much poorer substrates. A homology model of the human NPP1 was built based on the X-ray structure of its mouse orthologue. Docking studies were performed based on previously published mutagenesis data to rationalize the interactions of the different substrates and to explain the enzyme's preferences. The results provide an improved understanding of the interactions of NPP1 with its diverse substrates and will contribute to the validation of NPP1 as a drug target.     

Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification

The catabolism of ATP and other nucleotides participates partly in the important function of nucleotide salvage by activated cells and also in removal or de novo generation of compounds including ATP, ADP, and adenosine that stimulate purinergic signaling. Seven nucleotide pyrophosphatase/phosphodiesterase NPP family members have been identified to date. These isoenzymes, related by up conservation of catalytic domains and certain other modular domains, exert generally non-redundant functions via distinctions in substrates and/or cellular localization. But they share the capacity to hydrolyze phosphodiester or pyrophosphate bonds, though generally acting on distinct substrates that include nucleoside triphosphates, lysophospholipids and choline phosphate esters. PP_i generation from nucleoside triphosphates, catalyzed by NPP1 in tissues including cartilage, bone, and artery media smooth muscle cells, supports normal tissue extracellular PP_i levels. Balance in PP_i generation relative to PP_i degradation by pyrophosphatases holds extracellular PP_i levels in check. Moreover, physiologic levels of extracellular PP_i suppress hydroxyapatite crystal growth, but concurrently providing a reservoir for generation of pro-mineralizing P_i. Extracellular PP_i levels must be supported by cells in mineralization-competent tissues to prevent pathologic calcification. This support mechanism becomes dysregulated in aging cartilage, where extracellular PP_i excess, mediated in part by upregulated NPP1 expression stimulates calcification. PP_i generated by NPP1modulates not only hydroxyapatite crystal growth but also chondrogenesis and expression of the mineralization regulator osteopontin. This review pays particular attention to the role of NPP1-catalyzed PP_i generation in the pathogenesis of certain disorders associated with pathologic calcification.     

Rye germ acid phosphatase: properties of the enzyme and its activation by lectins

Acid phosphatase (EC 3.1.3.2) from rye germs is a glycoprotein of M, 90000 with subunit structure. The pH optimum for pNPP hydrolysis is 5.4. The best substrates for the enzyme are pNPP, PP_i and ATP. In the presence of plant lectins an increase in AcPase activity was found. ConA causes a 20% decrease of K_m^app and a 50% increase of V_max^app with pNPP as substrate.     

Structure–activity relationships in human RNA 3′-phosphate cyclase

RNA 3′-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2′,3′ cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP_i; transfer of AMP from Rtc1 to an RNA 3′-phosphate to form an RNA(3′)pp(5′)A intermediate; and attack by the terminal nucleoside O2′ on the 3′-phosphate to form an RNA 2′,3′ cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure–activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP α phosphorus is facilitated by proper orientation of the PP_i leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3′-phosphate terminated RNA, but not with an otherwise identical 3′-OH terminated RNA. Mutation of His320 had little impact on RNA 3′-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.     

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PP_i). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP_i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.     

Enzymatic characteristics of an ApaH-like phosphatase,PrpA, and a diadenosine tetraphosphate hydrolase,ApaH, from Myxococcus xanthus

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap₄A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards Ap_nA and ATP. In the presence of Mn²⁺, PrpA hydrolyzed Ap₄A into AMP and ATP, whereas in the presence of Co²⁺ PrpA hydrolyzed Ap₄A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards Ap_nA and ATP. Mn²⁺ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co²⁺ was required for Ap_nA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an Ap_nA hydrolase, respectively.     

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP_i). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2P_i). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP_i-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.     

The regulation of liver phosphoprotein phosphatase by inorganic pyrophosphate and cobalt.

The preincubation of rat liver crude extracts with ATP caused a 60% inactivation of phosphoprotein phosphatase in 30 min at 30 °C. The presence of Mg²⁺, or cyclic AMP, along with ATP in the preincubation mixture had no effect on the inactivation of phosphatase caused by ATP. The crude liver phosphatase was also inactivated by ADP or PP_i; PP_i being the most potent inactivating metabolite. AMP, adenosine or P_i were without any effect. The effect of ATP or PP_i was completely reversed by cobalt. The cobalt effect was very specific and could not be replaced by several metal ions tested except by Mn²⁺ which was partly active. With the aid of sucrose density gradient studies, it was also shown that PP_icauses an apparent conversion of a 4.1 S form to a 7.8 S form of the enzyme in rat liver extracts. Cobalt, on the other hand, converts the higher 7.8 S form to a lower 4.1 S form of the enzyme. The preincubation of purified rabbit liver phosphoprotein phosphatase with PP_i also caused a complete inactivation of the enzyme in 40 min. The inactivation of the enzyme by PP_i was completely reversed by cobalt. Unlike the apparent interconversion between different molecular forms of the enzyme by PP_i and cobalt in rat liver crude extracts, no such interconversion of purified rabbit liver phosphoprotein phosphatase was observed in the presence of PP_i and cobalt.     

Adenine nucleotide efflux in mitochondria induced by inorganic pyrophosphate

The efflux of mitochondrial adenine nucleotide which is induced by addition of PP_i to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 μM, V_max being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The K_m for PP_i of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PP_i.     

Sinomenine, theophylline, cysteine, and levamisole: Comparisons of their kinetic effects on mineral formation induced by matrix vesicles

The effects of sinomenine (SIN, an alkaloid extracted from the Chinese medicinal plant Sinomenium acutum used for centuries to treat rheumatic disease, including rheumatoid arthritis) on apatitic nucleation and matrix vesicle (MV)-induced mineral formation were compared with those of cysteine, levamisole, and theophylline. We found that SIN was not an inhibitor of tissue non-specific alkaline phosphatase (TNAP), a marker of biological mineralization, but confirmed that cysteine, levamisole, and theophylline were. Further, none of these four molecules directly affected the nucleation of hydroxyapatite (HA) formation, in contrast to pyrophosphate (PP_i) which did. Incubation of 0.25-1.0 mM cysteine, theophylline, or levamisole with MVs in synthetic cartilage lymph (SCL) containing AMP and Ca²⁺, but not inorganic phosphate (P_i), prolonged the induction time of mineral formation, apparently by inhibiting TNAP activity. SIN at the same levels neither inhibited TNAP activity nor affected the induction time of MV mineral formation. However, SIN did markedly delay MV-induced mineral formation in SCL containing P_i (instead of AMP) in a manner similar to theophylline, but to a lesser extent than levamisole. Cysteine did not delay, in fact it slightly accelerated MV-induced mineral formation in Pi-containing SCL. These findings suggest that levamisole, SIN and theophylline may directly affect Ca²⁺ and/or P_i accretion during mineral formation; however, TNAP was not directly involved. The possible roles of annexins and other ion transporters, such as proteins of the solute carrier family implicated in Ca²⁺ and P_i influx are discussed.     

Demonstration of ΔpH- and Δψ-induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum

It is possible to obtain synthesis of PP_i by artifical ion potentials in Rhodospirillum rubrum chromatophores. PP_i can be formed by K⁺-diffusion gradients (Δψ), H⁺ gradients (ΔpH) or a combination of both. In contrast, ATP can only be synthesized by imposed Δψ or Δψ+ΔpH. For ATP formation there is also a threshold value of K⁺ concentration below which synthesis of ATP is not possible. Such a threshold is not found for PP_i formation. Both PP_i and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PP_i can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PP_i is 200 nmol PP_i/μmol BChl, while that of ATP is no more than 25 nmol ATP/μmol BChl. The initial rates of syntheses were 0.50 μmol PP_i/μmol BChl per min and 2.0 μmol ATP/μmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.     

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K _m for p-Nph-5′-TMP hydrolysis of 61.8 ± 5.2 μM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5′-TMP hydrolysis with K _i values ranging 13–43 μM. Nucleotide analogs, α,β-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas α,β-metADP induced mixed inhibition, with K _i ranging from 2 to 20 μM. Chromatographic analysis revealed that α,β-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and α,β-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.     

Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores

Photosynthetic formation of inorganic pyrophosphate (PP_i) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PP_i synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PP_i synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PP_i synthesis much less. Even at low rates of electron transport a significant rate of PP_i synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PP_i synthesis is inhibited. It is shown that PP_i synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions.     

A new, convenient method for the rapid analysis of inorganic pyrophosphate.

A new method for the rapid analysis of inorganic pyrophosphate (PP_i) which utilizes the enzyme ATP sulfurylase is described. All components of the assay system are commercially available and inexpensive. The assay is linear over the range of 0.5–50.0 nmol of PP_i and is not affected by inorganic phosphate. ATP and PP_i can both be analyzed using this method.     

Dimethyl sulfoxide-induced hydroxyapatite formation: a biological model of matrix vesicle nucleation to screen inhibitors of mineralization

To elucidate the inhibition mechanisms of hydroxyapatite (HA), a biological model mimicking the mineralization process was developed. The addition of 4% (v/v) dimethyl sulfoxide (DMSO) in synthetic cartilage lymph (SCL) medium containing 2 mM calcium and 3.42 mM inorganic phosphate (P_i) at pH 7.6 and 37 °C produced HA as matrix vesicles (MVs) under physiological conditions. Such a model has the advantage of monitoring the HA nucleation process without interfering with other processes at the cellular or enzymatic level. Turbidity measurements allowed us to follow the process of nucleation, whereas infrared spectra and X-ray diffraction permitted us to identify HA. Mineral formation induced by DMSO and by MVs in the SCL medium produced crystalline HA in a similar manner. The nucleation model served to evaluate the inhibition effects of ATP, GTP, UTP, ADP, ADP-ribose, AMP, and pyrophosphate (PP_i). Here 10 μM PP_i, 100 μM nucleotide triphosphates (ATP, GTP, UTP), and 1 mM ADP inhibited HA formation directly, whereas 1 mM ADP-ribose and 1 mM AMP did not. This confirmed that the PP_i group is a potent inhibitor of HA formation. Increasing the PP_i concentration from 100 μM to 1 mM induced calcium pyrophosphate dihydrate. We propose that DMSO-induced HA formation could serve to screen putative inhibitors of mineral formation.     

Kinetic mechanism of the dimeric ATP sulfurylase from plants

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PP_i (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PP_i in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.     

Interaction of pyrophosphate with catalytic and noncatalytic sites of chloroplast ATP synthase

The effect of pyrophosphate (PP_i) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid membranes, PP_i competed with nucleotides for binding to noncatalytic sites. In the dark, PP_i was capable of tight binding to noncatalytic sites previously vacated by endogenous nucleotides, thereby preventing their subsequent interaction with ADP and ATP. The effect of PP_i on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PP_i inhibited ATPase activity of ATP synthase. Addition of PP_i to the reaction mixture at the step of preliminary illumination inhibited high initial activity of the enzyme, but stimulated its activity during prolonged incubation. These results indicate that the stimulating effect of PP_i light preincubation with thylakoid membranes on ATPase activity is caused by its binding to ATP synthase noncatalytic sites. The inhibition of ATP synthase results from competition between PP_i and ATP for binding to catalytic sites. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 956–962.     

Continuous pH stat assay technique for glutamine and asparagine synthetase enzyme systems, involving ATP conversion to ADP plus Pi and AMP plus PPi, respectively

Glutamine synthetase and asparagine synthetase systems with reactions involving lysis of ATP to ADP and P_i or AMP and PP_i are usually assayed by discontinuous sampling and analysis or by coupled enzymic systems. Experimental results confirm theoretical predictions that such reactions may be continuously and directly monitored by pH stat devices. Sample volumes of 0.5–1.0 ml and buret volumes of 0.05–0.25 ml, with ATP levels near 1 mm can be used routinely. The number of enzyme reactions involving ATP to which this technique can be applied is quite large.     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes

Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pH_v) of 4.74 ± 0.1 is generated in the presence of 0.1 millimolar PP_i, but only 4.93 ± 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PP_i. Cl⁻-stimulates the H⁺-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K_½ ≈ 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PP_i. The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pH_v unit has been estimated. From the free energy (−11.42 kilojoules per mole) for the hydrolysis of PP_i under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H⁺ per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H⁺-pump rather than as a pyrophosphate synthetase.