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PubMed1982

Purification and properties of inorganic pyrophosphatase of rat liver and hepatoma 3924A.

Inorganic pyrophosphatase (EC 3.6.1.1) has been purified to electrophoretic homogeneity from the soluble portion of the cytoplasm of rat Hepatoma 3924A and rat liver. It has a specific activity of 600 to 700 mumol inorganic orthophosphate liberated per min per mg protein at 25 degrees, a value in the same range as the highly purified enzymes from yeast and Escherichia coli. By all criteria applied, the hepatoma inorganic pyrophosphatase is identical with the liver enzyme. It is a dimer with subunits with molecular weights of approximately 30,000 to 33,000 and has a pH optimum of 7.4, a Km for pyrophosphate of 5 microM, and a Ka for Mg2+ of 0.3 mM with a pyrophosphate concentration of 0.2 mM. It is not inhibited by high Mg2+ concentrations up to 20 mM. Other metal ions such as Zn2+ and Ca2+ do not activate. Mn2+ activates to less than 10% that of Mg2+ at 0.6 mM and has no effect at 1 mM or higher. In the presence of optimal (4 mM) Mg2+ concentration, Ca2+, Mn2+, Hg2+, and F- at 0.2 mM inhibited strongly, but Zn2+ at 1 mM was not inhibitory. The enzyme had no phosphatase activity toward any of the purine or pyrimidine nucleoside mono-, di-, and triphosphates or toward p-nitrophenyl phosphate, beta-glycerophosphate, glucose 6-phosphate, or glucose 1-phosphate. Bromo- or iodoacetate at high concentration had no inhibitory effect, but p-chloromercuribenzoate and p-chloromercuriphenylsulfonate inhibited strongly at low concentration. The purified enzyme was very unstable but was protected markedly at or above the pH optimum of 7.4 by cysteine, dithiothreitol, and glutathione.

Adenosine Triphosphate↗

PubMed1982

[Cooperative mechanism of phosphorylation of the monomeric and dimeric forms of inorganic pyrophosphatase from baker's yeast].

A comparative study of phosphorylation of native dimeric and artificial monomeric forms of inorganic pyrophosphatase and its fluoride-stabilized complex with PPi has been carried out. The maximal incorporation of Pi for the dimeric and monomeric proteins is 0.5 and 1 mole per mole of subunit, respectively. The saturation kinetic curves are suggestive of strong positive cooperative interactions. The value of the Hill coefficient (5.5) for the free dimeric enzyme drastically changes upon the active center blockage and/or transition to the monomeric enzyme. Acceleration of dephosphorylation induced by Pi in the presence of Mg2+ is observed only in the case of the dimeric protein. The data obtained indicate that phosphorylation of native dimeric pyrophosphatase occurs according to a "flip-flop" mechanism; the Pi binding in the active center exerts a strong influence on individual steps of the reaction.

Binding Sites↗

PubMed1982

[Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase].

The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E. coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared. For both phosphatases the type of interaction was found to be independent on the nature of substrate. For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively. In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction. The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.

Acid Phosphatase↗

PubMed1983

[Effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a ubiquinone analog, and SH-reagents on the electrogenic function of pyrophosphatase from Rhodospirillum rubrum chromatophores].

Inorganic pyrophosphate-induced membrane potential generation in Rhodospirillum rubrum chromatophores is inhibited by 2,5-dibromo-2-methyl-6-isopropyl-p-benzoquinone (DBMIB). The inhibition is increased by menadione and dithionite and is not relieved by ascorbate; this effect is probably due to a displacement of the intrachromatophore quinones by DBMIB. The SH-reagents, N-ethylmaleimide and p-chloromercurybenzoate at high concentrations inhibit membrane potential generation driven by inorganic pyrophosphate hydrolysis, thus suggesting the involvement of SH-groups in pyrophosphatase operation. The mechanism of regulation of the mode of action of pyrophosphatase by quinones and the location of the SH-groups of the enzyme are discussed.

Bacterial Chromatophores↗

PubMed1983

[Two-stage mechanism of the fluoride inhibition of inorganic pyrophosphatase using the fluoride ion].

Some kinetic and spectral approaches have been used to study the interactions in the enzyme-Mg2+-F--pyrophosphate (or imidodiphosphate, a non-hydrolyzeable pyrophosphate analog) system underlying the mechanism of yeast inorganic pyrophosphatase inhibition by fluoride. The continuous curves of the enzymatic reaction were obtained with an automatic phosphate analyzer operating on the time scale of seconds. Increasing concentrations of NaF caused an increase in the inactivation rate constant to a constant level of 5.3 min-1 for PPi (pH 6.2-7.2) and 3.9 min-1 for imidodiphosphate, (pH 7.2). At a saturating fluoride concentration, the initial rate of PPi hydrolysis dropped to 10%. NaF and imidodiphosphate changed the protein spectrum at 270-310 nm and strengthened the binding of each other to the protein. The binding of F- required a Mg2+-binding site with Kd = 0.15 mM being filled in. The free enzyme and its Ca2+ complex did not bind F-. The experimental results indicate that pyrophosphatase inhibition by fluoride occurs in two steps. The inhibitor adds first to the Mg2+ ion on the enzyme in a readily reversible reaction causing a 90% decrease of the catalytic activity. Thereafter, a slow isomerization of the enzymesubstrate complex takes place, resulting in a complete loss of activity.

Calcium↗

PubMed1994

Purified vacuolar inorganic pyrophosphatase consisting of a 75-kDa polypeptide can pump H+ into reconstituted proteoliposomes.

Inorganic pyrophosphatase was purified to homogeneity from the vacuolar membranes of pumpkin hypocotyl tissue. The purified Inorganic pyrophosphatase consists of a single kind of 75-kDa polypeptide, and the stacked molecules with a repeating unit of 5.8 x 3.8 nm are seen under electron micrography. It exhibits H(+)-translocating activity across membranes coupled with PPi hydrolysis when it is reconstituted into proteoliposomes. A monovalent cation is required for the H+ translocation with the K+ ion being the most effective, but evidence for active transport of 42K+ into proteoliposomes was not obtained under the conditions tested. The hydrolysis of PPi by the reconstituted proteoliposomes is stimulated by the addition of a H+ ionophore, carbonyl cyanide p-trifluoromethyoxyphenylhydrazone, but not by a K+ ionophore, valinomycin. Both hydrolysis of PPi and PPi-dependent H+ translocation of the proteoliposomes are inhibited by N,N'-dicyclohexylcarbodiimide.

Amino Acid Sequence↗

PubMed1997

A hybrid mutant form of Escherichia coli inorganic pyrophosphatase.

The inorganic pyrophosphatase of Escherichia coli is a tightly hexamer of identical subunits. Upon interaction of its two mutant forms in which the trimer-trimer contacts were weakened because of E20D and H136Q substitutions, a hybrid hexameric E20D/H136Q-PPase is formed. The catalytic activity of its constituent H136Q trimer is same of its hexamer, whereas metal-binding affinity is significantly decreased. These results point to an interdependence of two trimers in catalysis by hexameric pyrophosphatase.

Binding Sites↗

PubMed1996

UDP-sugar pyrophosphatase of rat retina: subcellular localization and topography.

Rat retinal tissue possesses as a developmentally regulated, highly active pyrophosphatase activity that hydrolyzes UDP-GalNAc and UDP-Gal but not CMP-NeuAc (Martina et al.: J Neurochem 62:1274-1280, 1995). We show here that this activity, measured with UDP-[3H]GalNAc as substrate, is associated to the membrane fraction of rat retinal homogenates and, upon subfractionation by isopycnic centrifugation in sucrose density gradients, is concentrated in fractions enriched in light Golgi membranes. We examined also the topographic disposition of the catalytic site of the enzyme in the transverse plane of the membranes by measuring the effect of protease treatment and of added EDTA on its activity. Pronase inhibited 50% of the translocation of UDP-[3H]GalNAc to the lumen of the Golgi vesicles but did not affect the enzyme activity either in the absence or in the presence of detergent. EDTA, a membrane-impermeant molecule, inhibited 90% of the activity of the enzyme but did not affect translocation of UDP-[3H]GalNAc and inhibited only 25% the incorporation of [3H]GalNAc into endogenous glycoconjugates. These results indicate that the translocation of UDP-[3H]GalNAc was not necessary for hydrolysis to occur and strongly suggest that the catalytic site of the UDP-sugar pyrophosphatase is oriented toward the cytosolic side of the Golgi vesicles. We speculate that this activity limits the availability of UDP-GalNAc to its specific translocator and, consequently, the luminal concentration of the nucleotide in the Golgi vesicles. In this way, by limiting the availability of UDP-GalNAc for the conversion of GM3 to GM2 by the GM3:N-acetyl-galactosaminyl transferase, it would contribute to the preferential use of GM3 for synthesis of GD3 and other "b" pathway gangliosides that are characteristic of the rat retina.

Animals↗

PubMed1978

Inorganic pyrophosphatase activity of the synovial fluid. Kinetic and clinical study.

Inorganic pyrophosphatase activity has been partially characterized in joint fluid and determined in patients with sporadic and familial chondrocalcinosis and their controls. Optimal pH was established at 3.5 and Km values were estimated. Ca-2 and Mg-2 did not affect this activity whereas orthophosphate (Pi) strongly inhibited it. In the clinical study no significant differences were found among groups. This suggests that if a pyrophosphatase defect is present it might be localized in joint tissue and not be reflected in synovial fluid.

Adolescent↗

PubMed1985

Adenosine triphosphate pyrophosphohydrolase and neutral inorganic pyrophosphatase in pathologic joint fluids. Elevated pyrophosphohydrolase in calcium pyrophosphate dihydrate crystal deposition disease.

Adenosine triphosphate pyrophosphohydrolase (ATPPPH) and neutral inorganic pyrophosphatase activities were assayed in synovial fluids (SF) from 37 patients with a variety of arthropathies. ATPPPH activity was detected in all fluids, but was highest in patients with chronic chondrocalcinosis; its activity in patients with osteoarthritis was higher than that in patients with rheumatoid arthritis, gout, or pseudogout. ATPPPH activity correlated positively with SF pyrophosphate concentration and negatively with SF white blood cell count. Pyrophosphatase activity did not correlate with diagnosis, pyrophosphate level, or white blood cell count.

Adenosine Triphosphatases↗

PubMed1996

An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

Amino Acid Sequence↗

PubMed2006

Kinetic and structural properties of inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori.

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate (PPi) to orthophosphate (Pi) and controls the level of PPi in cells. PPase plays an essential role in energy conservation and provides the energy for many biosynthetic pathways. The Helicobacter pylori pyrophosphatase (HpPPase) gene was cloned, expressed, purified, and found to have a molecular weight of 20 kDa. The K(m) and V (max) of HpPPase were determined as 214.4 microM and 594 micromol Pi min(-1) mg(-1), respectively. PPi binds Mg(2+) to form a true substrate that activates the enzyme. However, free PPi could be a potent inhibitor for HpPPase. The effects of the inhibitors NaF, ATP, iminodiphosphate, and N-ethylmaleimide on HpPPase activity were evaluated. NaF showed the highest inhibition of the enzyme. Crystal structures of HpPPase and the PPi-HpPPase complex were determined. HpPPase comprises three alpha-helices and nine beta-strands and folds as a barrel structure. HpPPase forms a hexamer in both the solution and crystal states, and each monomer has its own PPi-binding site. The PPi binding does not cause a significant conformational change in the PPi-HpPPase complex, which might represent an inhibition state for HpPPase in the absence of a divalent metal ion.

Amino Acid Sequence↗

PubMed1995

Purification, cloning, and sequencing of archaebacterial pyrophosphatase from the extreme thermoacidophile Sulfolobus acidocaldarius.

Cytoplasmic pyrophosphatases are indispensible for the function of cellular bioenergetics. From the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius, situated at one of the lowest branches of the phylogenetic tree, a cytosolic pyrophosphatase has been isolated and purified 200-fold to electrophoretic homogeneity by combining ion-exchange and gel-exclusion chromatography. The native enzyme consists of a homotetramer of 71 kDa apparent molecular mass; the subunit displays an apparent molecular mass of 17 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an absolute requirement for divalent cations (Mg2+) and a temperature optimum of 75 degrees C coinciding with the growth optimum of the organism; the apparent estimated activation energy is 79.5 kJ/mol. A large variety of cytosolic extracts from other archaebacteria has been probed with a polyclonal antiserum raised against the purified protein; surprisingly, except for an extremely weak signal with S. solfataricus none of the other organisms showed any cross-reactivity. Also, Escherichia coli PPase does not cross-react. Based on N-terminal sequencing the gene has been cloned and sequenced. It codes for a 173-amino-acid protein with a calculated molecular mass of 19,365 kDa. Alignment with known eucaryotic and procaryotic PPases reveals invariant conservation of all residues presently assumed to be involved in metal and substrate binding. Unexpectedly, the highest similarity is found with the enzyme from the phylogenetically extremely distant eubacterium E. coli, but immunological cross-reactivity is absent. Similarity to the only known other archaebacterial PPase is much weaker. Using the 3D structure of the Thermus thermophilus enzyme as a scaffold an energy-minimized structural model is presented, deviating only minimally from the former. The structural features are discussed. The enzyme provides an excellent model for studies of thermostability and folding dynamics since heterologous overexpression has been achieved and genetically mutated forms become accessible.

Amino Acid Sequence↗

PubMed1993

Molecular cloning of cDNA for vacuolar membrane proton-translocating inorganic pyrophosphatase in Hordeum vulgare.

We obtained a cDNA clone(PP10) for the vacuolar membrane proton-translocating inorganic pyrophosphatase from barley roots by immunoscreening. The nucleotide sequence contained a 2308 bp open reading frame capable of coding for a polypeptide with 761 amino acids (M(r) 79,841). The polypeptide is highly hydrophobic and 12 membrane-spanning regions are deduced from the hydropathic evaluation. The characteristic cluster of the basic and acidic residues is observed in the hydrophilic segment. A consensus sequence with the dicyclohexyl-carbodiimide binding subunits of FOF1-type and vacuolar-type H(+)-ATPase is also observed in the membrane-spanning domain 5. Comparison of the deduced amino acid sequence with that of pyrophosphatase cDNA from Arabidopsis thaliana revealed 85.8% homology.

Amino Acid Sequence↗

PubMed2000

Cloning and characterization of the NADH pyrophosphatases from Caenorhabditis elegans and Saccharomyces cerevisiae, members of a Nudix hydrolase subfamily.

Two genes from Caenorhabditis elegans and Saccharomyces cerevisiae, coding for enzymes homologous to the Nudix hydrolase family of nucleotide pyrophosphatases, have been cloned and expressed in Escherichia coli. The purified enzymes are homodimers of 39.1 and 43. 5 kDa, respectively, are activated by Mg(2+) and Mn(2+), and are 30 to 50 times more active on NADH than on NAD(+). They both have a conserved array of amino acids downstream of the Nudix box first seen in the orthologous enzyme from E. coli which designates them as members of an NADH pyrophosphatase subfamily of the Nudix hydrolases.

Amino Acid Sequence↗

PubMed2001

An investigation of bisphosphonate inhibition of a vacuolar proton-pumping pyrophosphatase.

We report the results of a three-dimensional quantitative structure-activity relationship (3D-QSAR)/comparative molecular field analysis (CoMFA) of the activity of 18 bisphosphonates and imidodiphosphate in the inhibition of a mung bean (Vigna radiata L.) vacuolar proton pumping pyrophosphatase (V/H(+)-PPase; EC 3.6.1.1). We find an experimental versus QSAR predicted pK(app)(i) R(2) value of 0.89, a cross-validated R(2) = 0.77, and a bootstrapped R(2) = 0.89 for 18 bisphosphonates plus imidodiphosphate over the 1.3 microM to 425 microM range of K(app)(i) values. We also demonstrate that this approach has predictive utility (a 0.26 pK(app)(i) rms error for three test sets of 3 activity predictions each), corresponding to about a factor of two error in K(app)(i) prediction. The 3D-QSAR/CoMFA approach provides a quantitative method for predicting the activity of V/H(+)-PPase inhibitors and is likely to be of use in the design of novel pharmacological agents since all of the major human disease-causing parasitic protozoa contain large levels of pyrophosphate, together with V-type proton-pumping pyrophosphatases located in plant-like vacuoles (acidocalcisomes), which are absent in their mammalian hosts.

Diphosphonates↗

PubMed2001

The structures of Escherichia coli inorganic pyrophosphatase complexed with Ca(2+) or CaPP(i) at atomic resolution and their mechanistic implications.

Two structures of Escherichia coli soluble inorganic pyrophosphatase (EPPase) complexed with calcium pyrophosphate (CaPP(i)-EPPase) and with Ca(2+) (Ca(2+)-EPPase) have been solved at 1.2 and 1.1 A resolution, respectively. In the presence of Mg(2+), this enzyme cleaves pyrophosphate (PP(i)) into two molecules of orthophosphate (P(i)). This work has enabled us to locate PP(i) in the active site of the inorganic pyrophosphatases family in the presence of Ca(2+), which is an inhibitor of EPPase.Upon PP(i) binding, two Ca(2+) at M1 and M2 subsites move closer together and one of the liganded water molecules becomes bridging. The mutual location of PP(i) and the bridging water molecule in the presence of inhibitor cation is catalytically incompetent. To make a favourable PP(i) attack by this water molecule, modelling of a possible hydrolysable conformation of PP(i) in the CaPP(i)-EPPase active site has been performed. The reasons for Ca(2+) being the strong PPase inhibitor and the role in catalysis of each of four metal ions are the mechanistic aspects discussed on the basis of the structures described.

Binding Sites↗

PubMed2001

Expression in Escherichia coli of the thermostable inorganic pyrophosphatase from the Aquifex aeolicus and purification and characterization of the recombinant enzyme.

The gene encoding the inorganic pyrophosphatase from a hyperthermophilic bacterium, Aquifex aeolicus (Aae), was amplified by PCR. Then, the gene was overexpressed in Escherichia coli using a pJR-based expression plasmid, pAIPD. The recombinant Aae pyrophosphatase was purified 16.2-fold with a 53.4% yield and a specific activity of 34 U/mg protein by a combination of heating (to denature E. coli proteins) and two steps of DEAE-Sephacel column chromatography (nonabsorbed enzyme at pH 7.3 and absorbed enzyme at pH 8.0). This enzyme has an approximate molecular mass of 105,000 Da and consists of four subunits, each with a molecular mass of 24,500 Da. The enzyme shows the optimal activity in the pH range 7.5-8.0. The enzyme was stable at 80-95 degrees C. A divalent cation was absolutely required for the enzyme activity, Mg(2+) being most effective.

Bacteria↗