Pyrophosphate synthesis from phospho(enol)pyruvate catalyzed by precipitated magnesium phosphate with ”enzyme-like” activity

Summary The enzyme-like kinetic properties of precipitated magnesium phosphate as a catalyst for formation of pyrophosphate (PPi) from phospho (enol)pyruvate (PEP) are described. This synthesis occurs at a low temperature (37°C) and represents a model that may help us understand the relevance to chemical evolution of minerals as ancient catalysts whose functions could have been taken over by contemporary enzymes. An insoluble Pi.Mg matrix was formed in a medium with 80% of the water replaced by dimethyl sulfoxide as a way of simulating conditions in a drying pond. Phospho(enol)pyruvate adsorbs onto the Pi.Mg surface according to a Langmuir isotherm, and the PEP concentration dependence of PPi formation follows a Michaelian-like function. A yield of 33% for transformation of the initially adsorbed PEP into PPi was attained after 4 days of incubation with equimolecular concentrations of Pi, MgCl₂, and PEP. The magnesium concentration dependence for Pi and Mg precipitation, for adsorption of PEP onto solid Pi.Mg, and for PPi formation showed complex cooperative behavior. These results taken as a whole lead to the conclusion that the Pi.Mg surface not only provides a reactant for PPi formation but also catalyzes the reaction.Offprint requests to: A. Vieyra     

Model for prebiotic pyrophosphate formation: Condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents

Summary The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37°C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of³²PPi from³²Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.     

Two pathways of pyrophosphate hydrolysis and synthesis by yeast inorganic pyrophosphatase.

Initial rates of pyrophosphate hydrolysis and synthesis by baker's yeast inorganic pyrophosphatase and equilibrium amounts of enzyme-bound and free pyrophosphate were measured over wide ranges of Mg2+ and respective substrate concentrations. Computer analysis of these data, in conjunction with those on phosphate/water oxygen exchange [Kasho, V. N. & Baykov, A. A. (1989) Biochem. Biophys. Res. Comm. 161, 475-480], yielded values of the equilibrium constants for Mg2+ binding to free enzyme and central complexes and values of the forward and reverse rate constants for the four reaction steps, namely, PPi binding/release, PPi hydrolysis/synthesis and two Pi binding/release steps. All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg2+/PPi or 2 Pi bound. Product release is the slowest catalytic event in both hydrolysis and synthesis of pyrophosphate, at least, for the four-metal pathway. In the hydrolytic reaction, magnesium pyrophosphate binding is faster for the four-metal pathway, dissociation of the second Pi is faster for the three-metal pathway, while PPi hydrolysis and the release of the first Pi may proceed with similar rates. Release of pyrophosphate formed on the enzyme is faster for the three-metal pathway. Both pathways are expected to operate in vivo, and their relative contributions will vary with changes in the Mg2+ concentration, thus providing a means for pyrophosphatase-activity regulation.     

An assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling

A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated.     

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.     

Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum) chloroplasts was inhibited by inorganic pyrophosphate (PPi). Oxygen evolution was also inhibited by high concentrations of orthophosphate (Pi) and the inhibition was relieved by 3-phosphoglycerate. In contrast, the inhibition by PPi was not relieved by 3-phosphoglycerate, indicating that hydrolysis of PPi and accumulation of inhibitory concentrations of Pi were not occurring. In agreement with this suggestion, the percentage of (14)C-labeled products diffusing out of the chloroplasts was increased by Pi but not by PPi. The inhibition of O(2) evolution by PPi was reversed by ATP. The concentration of PPi required for 50% inhibition was 1.2 to 1.4 mm and the subsequent stimulation by ATP was half-maximal at 16 to 25 mum. Carbon dioxide-dependent O(2) evolution by spinach chloroplasts, or chloroplasts isolated from older pea plants, was not significantly inhibited by PPi.Chloroplasts were preloaded with (14)C-ATP and release of the labeled nucleotides was measured to assess the activity of adenine nucleotide transport across the inner chloroplast envelope membrane. A rapid exchange was promoted by the addition of exogenous ATP. Addition of PPi also resulted in a release of endogenous nucleotides. We suggest that PPi inhibits CO(2) fixation by entering the chloroplast in exchange for endogenous adenine nucleotides via the transporter on the inner envelope membrane. The subsequent depletion of the internal adenine nucleotide pool would result in decreased CO(2) fixation due to insufficient ATP. Addition of ATP to PPi-inhibited chloroplasts apparently results in uptake of catalytic amounts of ATP and restoration of the internal adenine nucleotide pool thus relieving the inhibition of CO(2) fixation.     

Population density and regulation of cell division in 3T3 cells. I. Inorganic phosphate levels, uptake and release.

Triggering mechanisms for initiating density dependent inhibition of cell division in 3T3 cell monolayers are activated approximately two to three population doublings prior to cessation of cell division at monolayer confluency. This activation occurs at a critical contact cell density of approximately 8 X 10(3) cells/cm2. During this period there are selective controls on transport and storage of required low molecular weight nutrients. A possible correlation between orthophosphate and rates of cell division has been investigated. We have demonstrated a relationship between cellular concentrations of orthophosphate and initiation of density dependent inhibition of cell division. Prior to critical intercellular contact, the [Pi] in 3T3 is 10 mM. During critical contact, this concentration is quickly reduced to approximately 2 mM and remains at this concentration to confluency. Similar alterations do not occur in Py 3T3 cells, which maintain a concentration of approximately 2 mM Pi regardless of cell density. After confluent 3T3 cells are released from inhibition of cell division the [Pi] must increase several-fold before DNA synthesis commences. These are physiological changes in 3T3 cellular [Pi] as a function of cell density, and cannot be attributed to nutrient depletion, altered transport of Pi into the cell, increased [ATP], or increased [PPi] levels. The controlled modulation of [Pi] may regulate glycolysis and coordinate counter-ion changes (Ca++) may regulate mitochondrial activity.     

ATP sulfurylase-dependent assays for inorganic pyrophosphate: applications to determining the equilibrium constant and reverse direction kinetics of the pyrophosphatase reaction, magnesium binding to orthophosphate, and unknown concentrations of pyrophosphate

A continuous, coupled, spectrophotometric assay is described in which the enzyme ATP sulfurylase is employed to measure the concentration of inorganic pyrophosphate (PPi) at equilibrium with known concentrations of inorganic orthophosphate (Pi) in the presence of excess inorganic pyrophosphatase (PPitase). In agreement with previous reports, the apparent equilibrium constant (Keq,app) of the PPi hydrolysis reaction was shown to decrease as the concentration of Mg2+ is increased. At pH 7.3, 30 degrees C, in the presence of 150 mM NaCl and 1 mM free Mg2+, Keq,app (calculated as [Pi]t2/[PPi]t) was 1950. Measurements of Keq,app at different total concentrations of Mg2+ and Pi permitted the determination of K0, the dissociation constant of the Mg-Pi complex. In 0.05 M Tris-Cl, pH 8.0, at 30 degrees C, K0 was 3.6 mM. In the presence of excess ATP sulfurylase, yeast PPitase catalyzed PPi formation from Pi with a specific activity (Vmax) of 9 units X mg protein-1 at pH 8.0, 30 degrees C, and 1 mM free Mg2+. Half-maximum reverse reaction velocity was observed at a total Pi concentration of 18 mM. (Under the same conditions, Vmax of the PPi hydrolysis reaction was 530 units X mg protein-1.) A radiochemical end point ("reaction-to-completion") assay for measuring unknown concentrations of PPi was devised. In the presence of excess 35S-adenosine-5'-phosphosulfate ([35S]APS) as the cosubstrate, 35SO2-4 formation was stoichiometric with added PPi. (The 35SO2-4 and [35S]APS are separated by adsorption of the latter onto charcoal.) The sensitivity of the assay can be adjusted by varying the specific radioactivity of the [35S]APS. In the absence of interfering substances, as little as 2 pmol of PPi per 1.0 ml assay volume can be measured. The sensitivity of the assay is reduced in the presence of ATP plus perchlorate (which synergistically inhibit the enzyme). However, if the bulk of the ATP is removed from perchloric acid extracts of tissues with glucose and hexokinase, initial intracellular levels as low as 1 microM can be measured. The possibility that most of the cellular PPi extracted with perchloric acid was originally enzyme bound is discussed.     

Two pathways for phosphate/water oxygen exchange by yeast inorganic pyrophosphatase

A scheme of Mg2+ and Pi binding to yeast inorganic pyrophosphatase has been deduced from the concentration dependencies of the rate of oxygen exchange between Pi and water. The exchange reaction requires the binding of MgPi and free Pi (pathway I) or two MgPi (pathway II) in addition to two Mg2+ ions bound in the absence of Pi. Pathway II predominates above 0.16 mM Mg2+. The rate of formation of bound PPi from bound Pi for pathway II is three times as high as that for pathway I. The results suggest that the binding of the fourth Mg2+ ion to pyrophosphatase stimulates its synthetic vs its hydrolytic capability.     

Detection and characterization of an additional site for binding of substrate and its analogs by inorganic pyrophosphatase

Phosphate, pyrophosphate, imidodiphosphate, EDTA and tripolyphosphate increase the rate constant for dissociation of the inorganic pyrophosphatase-substrate intermediate formed after cessation of the reaction by fluoride. The effect is enhanced in the given order 19-fold, the dependence of this effect on ligand concentration being hyperbolic. The values of the dissociation constants of the enzyme-ligand complexes lie within the concentration range of 0.16-1.0 mM. At high concentrations of Na2+ added simultaneously with the ligands this effect is decreased. The value of tau 1/2 for Pi binding to the enzyme-substrate compound is 0.15 min. The data obtained suggest that pyrophosphatase contains an anion ligand binding site, differing from that of the active one. This site does not affect the hydrolytic function of pyrophosphatase, as can be evidenced from the fact that Pi (9.5 mM) does not change the rate of enzymatic cleavage of PPi.     

Calcium, lanthanum, pyrophosphate, and hydroxyapatite: a comparative study in fibroblast mitogenicity

Calcium-containing crystals and elevated levels of calcium chloride (CaCl2) and lanthanum chloride (LaCl3) have been previously reported to enhance the proliferative activity of cultured fibroblasts. We have investigated the relative mitogenicity of these agents, whether they function via precipitation on the cell surface and whether they interact with one another. Confluent cultures of newborn foreskin fibroblasts provided with fresh medium containing 10% fetal bovine serum (FBS) in the presence of hydroxyapatite (HA), pyrophosphate (PPi), LaCl3 (La), or additional CaCl2 (Ca) were all stimulated more than control cultures provided with fresh medium and 10% FBS alone as assessed by cell counts 5 days later. Increases in cell yield above the original confluent cell density were 316% for La, 271% for Ca, 189% for HA, 131% for PPi, and 45% for controls. Addition of fresh medium containing 10% FBS and epidermal growth factor or fresh medium containing 20% FBS as additional points of reference yielded increases of 204 and 107%, respectively, over original confluent density. Stimulation induced by La or Ca was significantly greater (P less than 0.001) than the stimulation induced by each of the other treatments. The same treatments added to confluent cultures without a change of medium also renewed mitotic activity, with La and Ca again the most mitogenic and approximately doubling the pretreatment cell yields. Cultures incubated in an inverted position to avoid cell contact with precipitates in the medium were also stimulated by La and Ca, but not by HA and PPi. When added to confluent cultures simultaneously supplemented with optimal additional Ca, La decreased Day 5 cell yields in a dose-dependent manner at low concentrations (0.03-0.2 mM) but increased cell yields over those obtained with 0.2 mM LaCl3 again in a dose-dependent manner at higher concentrations. Thus, while HA and PPi act via precipitation on the cell surface, the more mitogenic agents La and Ca function in solution and appear to stimulate cell division by different nonadditive mechanisms. These findings suggest multiple mechanisms of membrane participation in mitogen responsiveness and in density-dependent inhibition of growth.     

Proton transport in maize tonoplasts supported by fructose-1,6-bisphosphate cleavage. Pyrophosphate-dependent phosphofructokinase as a pyrophosphate-regenerating system

The energy derived from pyrophosphate (PPi) hydrolysis is used to pump protons across the tonoplast membrane, thus forming a proton gradient. In a plant's cytosol, the concentration of PPi varies between 10 and 800 microm, and the PPi concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H+-pyrophosphatase is 30 microm. In this report, we show that the H+-pyrophosphatase of maize root vacuoles is able to hydrolyze PPi (Reaction 2) formed by Reaction 1, which is catalyzed by PPi-dependent phosphofructokinase (PFP): Fructose-1,6-bisphosphate (F1,6BP) + Pi <--> PPi +Fructose-6-phosphate (F6 P) (reaction 1) PPi --> 2 Pi (reaction 2) H+cyt --> H+vac (reaction 3) F1,6BP + H+cyt <--> H+vac + F6P + Pi (reaction 4) During the steady state, one-half of the inorganic phosphate released (Reaction 4) is ultimately derived from F1,6BP, whereas PFP continuously regenerates the pyrophosphate (PPi) hydrolyzed. A proton gradient (DeltapH) can be built up in tonoplast vesicles using PFP as a PPi-regenerating system. The Delta pH formed by the H+-pyrophosphatase can be dissipated by addition of 20 mm F6P, which drives Reaction 1 to the left and decreases the PPi available for the H+-pyrophosphatase. The maximal Delta pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found in higher plants tissues.     

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.     

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.     

Use of phosphate to avoid uranium toxicity in Arabidopsis thaliana leads to alterations of morphological and physiological responses regulated by phosphate availability

Uranium is an ubiquitous pollutant with known chemical and radiological toxicity, which is naturally present in the plant environment. Due to its high affinity for phosphate, insoluble uranium-phosphate precipitates are formed in soils as well as in contaminated plant cells. To date, consequences of such interactions on uranium toxicity and on phosphate availability and metabolism in plants are unknown. This study aims at evaluating in which extent uranium-phosphate interactions have an effect on physiological and molecular mechanisms involved in plant responses (i) to uranium contamination and (ii) to phosphate availability in Arabidopsis thaliana.Inorganic phosphate (Pi) supply in U-contaminated medium was shown to decrease U bioaccumulation and U toxic effects on plant biomass and root cell viability. Besides, U was shown to disturb plant responses to Pi availability. Indeed, in Pi-sufficient conditions, high U concentrations promoted the induction of phosphate starvation responses in plants. However, the most drastic effects have been observed in Pi-deficient conditions as U affected the following plant responses to Pi-starvation: root architecture modulation, phosphate acquisition and optimization of phosphate allocation. Indeed, despite the low Pi status of these plants, 2 μM U inhibited the primary root growth arrest normally triggered by low Pi. Moreover, Pi uptake and translocation to shoot were reduced. The root concentration of soluble inorganic phosphate decreased in Pi-starved plantlets contaminated with U, despite the enhancement of shoot-to-root remobilization of Pi. The observations of intracellular and apoplastic deposits of U and P in roots using electron microscopy (TEM-EDX) and secondary ion mass spectroscopy (NanoSIMS) provided evidence that Pi flux disturbance is a consequence of the use of Pi to immobilize U within roots.     

Theoretical analysis of distribution of [18O]Pi species during exchange with water. Application to exchanges catalyzed by yeast inorganic pyrophosphatase

A theoretical analysis has been derived which allows the analytical calculation of the complete distribution of 18O-labeled Pi species expected to occur during medium Pi equilibrium HOH exchange of [18O]Pi and to be produced by intermediate Pi equilibrium HOH exchange during net hydrolysis of [18O]PPi or other labeled phosphate compounds. The observed distributions with catalysis by yeast inorganic pyrophosphatase are found to agree closely with the theoretical values indicating that the exchange reaction can be adequately described by a unique value of the partitioning of bound Pi between release from the enzyme versus formation of bound PPi with loss of an oxygen to the water. The limitations on the exclusion of other mechanisms are discussed. The extent of this partitioning does change, however, under some experimental conditions. At low pH, with activation by Mg2+ or Mn2+, the relative rate of release of Pi is found to increase. The extent of exchange is also dependent on the nature of the activating metal, being greatest with Co2+. During PPi hydrolysis with PPi in excess over Mg2+, a shift to lower extents of exchange is observed.     

Characteristics of the formation of enzyme-bound ATP from medium inorganic phosphate by mitochondrial F1 adenosinetriphosphatase in the presence of dimethyl sulfoxide

Addition of dimethyl sulfoxide promotes the formation of enzyme-bound ATP from medium Pi by mitochondrial F1 adenosinetriphosphatase that has tightly bound ADP present. Measurements are reported of medium Pi in equilibrium H18OH exchange and of the dependence of formation of enzyme-bound ATP on Pi concentration. Attainment of an apparent equilibrium between medium Pi and bound ATP requires longer than 30 min, even though the rates of Pi binding and release after apparent equilibrium is reached would suffice for a faster approach to equilibrium. Slow protein conformational changes or other unknown modulating factors may be responsible for the slow rate of bound ATP formation. After apparent equilibrium is reached, each Pi that binds to the enzyme reversibly forms ATP about 50 times before being released to the medium. The rate of interconversion of bound ATP to bound ADP and Pi is much slower than that in the absence of dimethyl sulfoxide as measured with sufficiently low ATP concentrations so that single-site catalysis is favored. Although the interconversion rate is slowed, the equilibrium constant for bound ATP formation from bound ADP and Pi is not far from unity. Dimethyl sulfoxide favors the formation of enzyme-bound ATP by promoting the competent binding of Pi to enzyme with ADP bound at a catalytic site rather than by promoting formation of bound ATP from bound ADP and Pi.