Submitochondrial localization and function of alkaline inorganic pyrophosphatase in maize seedlings.

Alkaline inorganic pyrophosphatase and Mg-ATPase are localized within the mitoplast of maize seeding mitochondria. NaF inhibited the PPase activity, whereas oligomycin and dicyclohexylcarbodiimide inhibited the Mg-ATPase activity. The mitoplast preparation synthesized PPi from Pi under conditions excluding hydrolysis of endogenous ATP. PPi synthesis was inhibited by ADP, antimycin A, NaCN and 2,4- dinitrophenol but not by oligomycin. It is suggested that PPi synthesis in the maize seedling mitochondria proceeds at the expense of the energy of electron transport chain and is independent of the ATP synthesis.     

Inorganic pyrophosphatase activity in cell-free extracts of Ureaplasma urealyticum

Cell-free extracts of Ureaplasma urealyticum strains Pi and T960 (CX8) (serovars 6 and 8, respectively) metabolized inorganic pyrophosphate (PPi). The inorganic pyrophosphatase (PPase) activity was greatest with Mg2+ as cofactor, but Mn2+ acted as a poor substitute. The PPases of the two serovars differed electrophoretically. Although the highest PPase activity was obtained using PPi as substrate, the enzyme could also utilize to a lesser degree both tripolyphosphate and trimetaphosphate. No activity was observed against beta-glycerophosphate, naphthyl phosphates, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, thiamin pyrophosphate, phosphoribosylpyrophosphate, ADP or ATP. Acid- and alkaline-phosphatase activities were observed with naphthyl phosphates as substrates, but they did not have the same electrophoretic mobility on gels as the PPase activity. U. urealyticum PPase was inhibited by oxidized glutathione, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, p-chloromercuribenzoic acid, Mn2+, Zn2+ and Ca2+. Neither reduced glutathione, L-cysteine nor Co2+ enhanced activity. PPi can act as a substrate or regulator of certain metabolic reactions, and PPi metabolism can function in bacterial bioenergetics; its role in ureaplasmas is presently unclear.     

PYP-1, inorganic pyrophosphatase, is required for larval development and intestinal function in C. elegans

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into phosphate (Pi), which provides a thermodynamic driving force for important biosynthetic reactions. The nematode Caenorhabditis elegans gene C47E12.4 encodes a PPase (PYP-1) which shows 54% amino acid identity with human PPase. PYP-1 exhibits specific enzyme activity and is mainly expressed in the intestinal and nervous system. A null mutant of pyp-1 reveals a developmental arrest at early larval stages and exhibits gross defects in intestinal morphology and function. The larval arrest phenotype was successfully rescued by reintroduction of the pyp-1 gene, suggesting that PYP-1 is required for larval development and intestinal function in C. elegans.     

Investigation of the regiospecificity and stereospecificity of proton transfer in the yeast inorganic pyrophosphatase catalyzed reaction

The regiospecificity and stereospecificity of proton transfer in the yeast inorganic pyrophosphatase (PPase) catalyzed hydrolysis of P1,P2-bidentate Mg(H2O)4(PPi)2- were probed with exchange-inert metal complexes of imidodiphosphate (PNP) and thiopyrophosphate (PPS). PPase was unable to catalyze the hydrolysis of Mg(H2O)4PNP and P1,P2-bidentate Co(NH3)4PNP under conditions that resulted in rapid hydrolysis of the corresponding metal-PPi complexes. PPase was found to catalyze the hydrolysis of Mg(H2O)4PPS at 17% the rate of Mg(H2O)4PPi hydrolysis. The Km of Mg(H2O)4PPS was determined to be 300 microM, which is a value 10-fold greater than that observed for Mg(H2O)4PPi. P1,P2-Bidentate Cr(H2O)4PPS and Co(NH3)4PPS (prepared from PPS) were both found to be substrates for PPase. The enzyme specifically catalyzed the hydrolysis of the Rp enantiomers of these complexes and not the Sp enantiomers. These results are accommodated by a reaction mechanism involving enzyme-mediated proton transfer to the pro-R oxygen atom of the incipient phosphoryl leaving group of the bound P1,P2-bidentate Mg(H2O)4PPi2- complex.     

Effects of spermine and spermidine on the inorganic pyrophosphatase of Streptococcus faecalis. Interactions between polyamines and inorganic pyrophosphate.

We have shown a dual role for Mg2+ in the hydrolysis of PPi catalysed by inorganic pyrophosphatase (PPase; EC 3.6.1.1) of Streptococcus faecalis; Mg2+ is necessary for the formation of the substrates, Mg1PPi2- and Mg2PPi0, and it also acts as an allosteric activator [Lahti + Jokinen (1985) Biochemistry 24, 3526-3530]. No activity can be observed with S. faecalis PPase in the absence of bivalent cations, which indicates that free PPi cannot serve as a substrate for this enzyme. However, significant activities were observed in the presence of spermine and spermidine, even though no bivalent cations were present. It was shown by particle-induced gamma-ray emission and particle-induced X-ray-emission analysis that the polyamines used were not contaminated with Mg2+ or any other bivalent cations that could support PPase activity. Hence it is obvious that polyamines are able to form a complex with PPi that serves as a substrate for PPase. The apparent stability constants for the 1:1 adducts of spermine and spermidine were estimated by a resin competition method. The values obtained at pH 7.5 were 2.7 X 10(3) M-1 and 6.4 X 10(2) M-1 respectively. Kinetic results further suggested that polyamines can also substitute for Mg2+ as an activator in vitro. The physiological significance of these polyamine effects were discussed.     

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.     

植物焦磷酸酶(PPase)的研究进展

植物焦磷酸酶(PPase)可分为存在于细胞质中可溶性的无机焦磷酸酶和与膜结合的不可溶性焦磷酸酶.后者不仅能水解焦磷酸,同时还具有质子泵的功能.橡胶树乳管中与黄色体膜结合的不可溶性焦磷酸酶,是调控橡胶生物合成的一个必不可少的酶.对植物焦磷酸酶的结构及其功能和分子生物学研究的进展进行了综合论述,并着重阐述了焦磷酸酶在橡胶树橡胶生物合成中的作用.     

Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites.

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli-expressed recombinant enzyme has a specific activity of 937 micro mol Pi.min-1.mg-1 protein corresponding to a kcat value of 638 s-1 at 55 degrees C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.     

The role of lipids in the regulation of ATP and PPi synthesis in mitochondria

It was demonstrated previously that mitochondria of higher and lower eukaryotes can synthesize, in the course of oxidative phosphorylation, not only ATP but also inorganic pyrophosphate (PPi). Two PPases were isolated from bovine heart mitochondria (soluble--PPase I and membrane--PPase II). Coupling PPase II, in contrast to PPase I, contains phosphatidyl choline, but PPase I is lipidized readily in the presence of different phospholipids. Reconstitution experiments of the PPi synthesis system have shown that after lipidization PPase I is able to incorporate into submitochondrial particles (SMP) and becomes a coupling factor for oxidation and PPi synthesis. It seems that phospholipid is indispensible for incorporation into the membrane and the manifestation of the coupling activity of the enzyme. The effect of lipids on the activity of soluble and membrane-bound pyrophosphatase was studied. It is shown that PPase II phospholipid is involved in the regulation of the hydrolase activity of the isolated enzyme. However, hydrolysis of PPi by SMP and its synthesis by mitochondria are affected by cooperative rearrangements of the entire lipid component of the membrane rather than by changes in the phase state of phosphatidyl choline contained in PPase II. An opposite response of ATP and PPi synthesis to changes in viscosity makes it likely that the viscosity of the mitochondrial inner membrane may control the levelling of these two processes in mitochondria.     

The quaternary structure of Escherichia coli inorganic pyrophosphatase is essential for phosphorylation

The hexameric inorganic pyrophosphatase (PPase) is irreversibly inactivated by phosphoric acid monoesters. The inactivation kinetics are consistent with the formation of a dissociable complex of the phosphoric acid monoester with the enzyme, followed by phosphorylation of the dicarboxylic amino acid of its active site. PPi and its analogues, binding at the regulatory site, release the inhibitor from the active site and thus restore PPase activity. Chemically identical subunits in the hexameric PPase interact, promoting their cooperativity in a reaction with phosphoric acid monoesters. The trimeric and monomeric PPase, exhibiting full catalytic activity, form a dissociable complex with the phosphoric acid monoesters but, in contrast to the hexameric PPase, do not form a covalent bond with them. This indicates that the native hexameric structure is essential for the irreversible inactivation of Escherichia coli PPase by phosphoric acid monoesters. Possible nontraditional pathways for activity regulation of PPase are discussed.     

Determination of the inorganic pyrophosphate level and its subcellular localization in Chara corallina

In order to determine the concentration of pyrophosphate (PPi) and its subcellular distribution in Chara corallina, a new method to concentrate PPi from cell extracts was developed. PPi was extracted and concentrated as Ca2P2O7 under alkaline conditions. The amount of PPi in the precipitate was measured using an enzyme system containing pyrophosphate:fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) coupled to NADH oxidation in the presence of [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid. The subcellular localization of PPi and inorganic phosphate (Pi) was studied using the intracellular perfusion technique. The relative volumes of the cytoplasm (6.4%) and the vacuole (93.6%) were determined by perfusing Lucifer Yellow CH into the vacuole and by assuming that the Lucifer Yellow CH dead space represented the cytoplasmic volume. The volume of the chloroplast layer was determined microscopically, and it was found that it occupied 10% of the Chara cytoplasm. PPi was present predominantly in the cytosol at a level of 193 microM, while it existed in the vacuole at a level of only 2.20 microM and less than 1 microM in chloroplasts. By contrast, Pi was distributed almost equally in the cytosol (12.0 mM), chloroplasts (16.2 mM), and the vacuole (6.70 mM). The electrochemical potential gradient across the tonoplast for H+ (delta mu H+ = -11.6 to -18.0 KJ/mol) was nearly equal to the free energy release from the hydrolysis of PPi in cytoplasm (delta Gpp = -18.9 KJ/mol), indicating that the H+-translocating inorganic pyrophosphatase can work as a H+ pump in C. corallina.     

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.     

Method for real-time detection of inorganic pyrophosphatase activity

A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.     

Links Between Hydrothermal Environments,Pyrophosphate, Na<Superscript>+</Superscript>, and Early Evolution

The discovery that photosynthetic bacterial membrane-bound inorganic pyrophosphatase (PPase) catalyzed light-induced phosphorylation of orthophosphate (Pi) to pyrophosphate (PPi) and the capability of PPi to drive energy requiring dark reactions supported PPi as a possible early alternative to ATP. Like the proton-pumping ATPase, the corresponding membrane-bound PPase also is a H⁺-pump, and like the Na⁺-pumping ATPase, it can be a Na⁺-pump, both in archaeal and bacterial membranes. We suggest that PPi and Na⁺ transport preceded ATP and H⁺ transport in association with geochemistry of the Earth at the time of the origin and early evolution of life. Life may have started in connection with early plate tectonic processes coupled to alkaline hydrothermal activity. A hydrothermal environment in which Na⁺ is abundant exists in sediment-starved subduction zones, like the Mariana forearc in the W Pacific Ocean. It is considered to mimic the Archean Earth. The forearc pore fluids have a pH up to 12.6, a Na⁺-concentration of 0.7 mol/kg seawater. PPi could have been formed during early subduction of oceanic lithosphere by dehydration of protonated orthophosphates. A key to PPi formation in these geological environments is a low local activity of water.     

Alternative photophosphorylation,inorganic pyrophosphate synthase and inorganic pyrophosphate

This minireview in memory of Daniel I. Arnon, pioneer in photosynthesis research, concerns properties of the first and still only known alternative photophosphorylation system, with respect to the primary phosphorylated end product formed. The alternative to adenosine triphosphate (ATP), inorganic pyrophosphate (PPi), was produced in light, in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum, when no adenosine diphosphate (ADP) had been added to the reaction mixture (Baltscheffsky H et al. (1966) Science 153: 1120–1122). This production of PPi and its capability to drive energy requiring reactions depend on the activity of a membrane bound inorganic pyrophosphatase (PPase) (Baltscheffsky M et al. (1966) Brookhaven Symposia in Biology, No. 19, pp 246–253); (Baltscheffsky M (1967) Nature 216: 241–243), which pumps protons (Moyle J et al. (1972) FEBS Lett 23: 233–236). Both enzyme and substrate in the PPase (PPi synthase) are much less complex than in the case of the corresponding adenosine triphosphatase (ATPase, ATP synthase). Whereas an artificially induced proton gradient alone can drive the synthesis of PPi, both a proton gradient and a membrane potential are required for obtaining ATP. The photobacterial, integrally membrane bound PPi synthase shows immunological cross reaction with membrane bound PPases from plant vacuoles (Nore BF et al. (1991) Biochem Biophys Res Commun 181: 962–967). With antibodies against the purified PPi synthase clones of its gene have been obtained and are currently being sequenced. Further structural information about the PPi synthase may serve to elucidate also fundamental mechanisms of electron transport coupled phosphorylation. The existence of the PPi synthase is in line with the assumption that PPi may have preceded ATP as energy carrier between energy yielding and energy requiring reactions.     

Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3

A gene encoding for a putative Family I inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 degrees C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 degrees C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05-0.5 mM). The K(m) for pyrophosphate and Mg2+ were 113 and 303 microM, respectively; and maximum velocity, V(max), was estimated at 930 U mg(-1).     

Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.     

Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions

Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration.     

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.     

Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells.

No correlation was observed between the level of inorganic pyrophosphatase (PPase) and the intracellular concentration of PPi in Escherichia coli cells. In exponentially growing cells the intracellular PPi concentration was in every case 1.5 nmol/mg (dry weight) or about 0.5 mM, even though the amount of PPase was varied from 15 to 2,600% of the control amount by mutation or by using a multicopy plasmid with an inserted gene (ppa) encoding PPase. The PPi concentration could, however, be increased or decreased from the control level under some stressful conditions.