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European Journal of Biochemistry Jun 1999Cell surface ATPases (ecto-ATPases or E-ATPases) hydrolyze extracellular ATP and other nucleotides. Regulation of extracellular nucleotide concentration is one of their...
Cell surface ATPases (ecto-ATPases or E-ATPases) hydrolyze extracellular ATP and other nucleotides. Regulation of extracellular nucleotide concentration is one of their major proposed functions. Based on enzymatic characterization, the E-ATPases have been divided into two subfamilies, ecto-ATPases and ecto-ATP-diphosphohydrolases (ecto-ATPDases). In the presence of either Mg2+ or Ca2+, ecto-ATPDases, including proteins closely related to CD39, hydrolyze nucleoside diphosphates in addition to nucleoside triphosphates and are inhibited by millimolar concentrations of azide, whereas ecto-ATPases appear to lack these two properties. This report presents the first systematic kinetic study of a purified ecto-ATPDase, the chicken oviduct ecto-ATPDase (Strobel, R.S., Nagy, A.K., Knowles, A.F., Buegel, J. & Rosenberg, M.O. (1996) J. Biol. Chem. 271, 16323-16331), with respect to ATP and ADP, and azide inhibition. Km values for ATP obtained at pH 6.4 and 7.4 are 10-30 times lower than for ADP and the catalytic efficiency is greater with ATP as the substrate. The enzyme also exhibits complicated behavior toward azide. Variable inhibition by azide is observed depending on nucleotide substrate, divalent ion, and pH. Nearly complete inhibition by 5 mm azide is obtained when MgADP is the substrate and when assays are conducted at pH 6-6.4. Azide inhibition diminishes when ATP is the substrate, Ca2+ as the activating ion, and at higher pH. The greater efficacy of azide in inhibiting ADP hydrolysis compared to ATP hydrolysis may be related to the different modes of inhibition with the two nucleotide substrates. While azide decreases both Vmax and Km for ADP, it does not alter the Km for ATP. These results suggest that the apparent affinity of azide for the E.ADP complex is significantly greater than that for the free enzyme or E.ATP. The response of the enzyme to three other inhibitors, fluoride, vanadate, and pyrophosphate, is also dependent on substrate and pH. Taken together, these results are indicative of a discrimination between ADP and ATP by the enzyme. A mechanism of azide inhibition is proposed.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Apyrase; Azides; Chickens; Diphosphates; Enzyme Inhibitors; Fluorides; Hydrogen-Ion Concentration; Hydrolysis; Oviducts; Vanadates
PubMed: 10336618
DOI: 10.1046/j.1432-1327.1999.00389.x -
Annals of the Rheumatic Diseases Apr 1991Deposition of intra-articular calcium pyrophosphate is associated with both aging and arthropathy; increased concentrations of free pyrophosphate (PPi) may contribute to... (Comparative Study)
Comparative Study
Deposition of intra-articular calcium pyrophosphate is associated with both aging and arthropathy; increased concentrations of free pyrophosphate (PPi) may contribute to such deposition. Free pyrophosphate and nucleoside triphosphate pyrophosphatase (NTPase) were estimated in synovial fluids from 50 subjects with normal knees and from 44 patients with rheumatoid arthritis, 61 with pyrophosphate arthropathy, and 59 with osteoarthritis. For arthropathic knees clinically assessed inflammation was classified as active or inactive using a summated score of six clinical features. The order of PPi (mumol/l) and NTPase (mumol PPi/30 min/mg protein) was pyrophosphate arthropathy greater than osteoarthritis greater than rheumatoid arthritis (median PPi, NTPase respectively: for pyrophosphate arthropathy 15.9, 0.45; for osteoarthritis 9.3, 0.25; for rheumatoid arthritis 4.4, 0.18), with significant differences between all groups. In pyrophosphate arthropathy both PPi (mumol/l) and NTPase (mumol PPi/30 min/mg protein) were higher than normal (15.9, 0.45 v 8.6, 0.2 respectively), but findings in osteoarthritis did not differ from normal. The inflammatory state of the knee had a distinct but variable effect on synovial fluid findings in rheumatoid arthritis and pyrophosphate arthropathy, but not in osteoarthritis. There was no correlation of either PPi or NTPase with age, or between PPi and NTPase in any group. This study provides in vivo data for synovial fluid PPi and NTPase. It suggests that factors other than PPi need to be considered in a study of crystal associated arthropathy. Clinical inflammation, as well as diagnosis, is important in synovial fluid studies.
Topics: Adult; Aged; Aged, 80 and over; Arthritis; Arthritis, Rheumatoid; Calcium Pyrophosphate; Diphosphates; Female; Humans; Joint Diseases; Knee Joint; Male; Middle Aged; Osteoarthritis; Pyrophosphatases; Synovial Fluid
PubMed: 1851409
DOI: 10.1136/ard.50.4.214 -
Chemical Communications (Cambridge,... Apr 2011Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have...
Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have been suggested to largely control their reactions via steric effects, serving as templates. However, recent results highlight electrostatic effects also exerted by these enzymes. Perhaps not surprisingly, the pyrophosphate co-product released in the initiating and rate-limiting chemical step provides an obvious counter-ion that may steer carbocation migration towards itself. This is emphasized by the striking effects of a recently uncovered single residue switch for diterpene synthase product outcome, whereby substitution of hydroxyl residues for particular aliphatic residues has been shown to be sufficient to "short-circuit" complex cyclization and/or rearrangement reactions, with the converse change further found to be sufficient to increase reaction complexity. The mechanistic hypothesis for the observed effects is hydroxyl dipole stabilization of the specific carbocation formed by initial cyclization, enabling deprotonation of this early intermediate, whereas the lack of such stabilization (i.e. in the presence of an aliphatic side chain) leads to carbocation migration towards the pyrophosphate co-product, resulting in a more complex reaction. This is further consistent with the greater synergy exhibited between pyrophosphate and aza-analogs of late, relative to early, stage carbocation intermediates, and crystallographic analysis of the monoterpene cyclase bornyl diphosphate synthase wherein mechanistically non-relevant counter-ion pairing between aza-analogs of early stage carbocation intermediates and pyrophosphate is observed. Thus, (di)terpene synthases seem to mediate specific reaction outcomes, at least in part, by providing electrostatic effects to counteract those exerted by the pyrophosphate co-product.
Topics: Alkyl and Aryl Transferases; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Cyclization; Diphosphates; Diterpenes; Kinetics; Static Electricity; Stereoisomerism
PubMed: 21305070
DOI: 10.1039/c0cc02960b -
Biochemical and Biophysical Research... Mar 2013Ap4A hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17), an enzyme involved in a number of biological processes, is characterized as cleaving the...
Ap4A hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17), an enzyme involved in a number of biological processes, is characterized as cleaving the polyphosphate chain at the fourth phosphate from the bound adenosine moiety. This paper presents the crystal structure of wild-type and E58A mutant human Ap4A hydrolase. Similar to the canonical Nudix fold, human Ap4A hydrolase shows the common αβα-sandwich architecture. Interestingly, two sulfate ions and one diphosphate coordinated with some conserved residues were observed in the active cleft, which affords a better understanding of a possible mode of substrate binding.
Topics: Acid Anhydride Hydrolases; Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Diphosphates; Humans; Molecular Sequence Data; Mutant Proteins; Mutation; Phosphoric Monoester Hydrolases; Protein Structure, Secondary; Sulfuric Acid Esters
PubMed: 23384440
DOI: 10.1016/j.bbrc.2013.01.095 -
Acta Crystallographica. Section F,... Dec 2017The exosporium layer of Bacillus anthracis spores is rich in L-rhamnose, a common bacterial cell-wall component, which often contributes to the virulence of pathogens by...
The exosporium layer of Bacillus anthracis spores is rich in L-rhamnose, a common bacterial cell-wall component, which often contributes to the virulence of pathogens by increasing their adherence and immune evasion. The biosynthetic pathway used to form the activated L-rhamnose donor dTDP-L-rhamnose consists of four enzymes (RfbA, RfbB, RfbC and RfbD) and is an attractive drug target because there are no homologs in mammals. It was found that co-purifying and screening RfbC (dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase) from B. anthracis in the presence of the other three B. anthracis enzymes of the biosynthetic pathway yielded crystals that were suitable for data collection. RfbC crystallized as a dimer and its structure was determined at 1.63 Å resolution. Two different ligands were bound in the protein structure: pyrophosphate in the active site of one monomer and dTDP in the other monomer. A structural comparison with RfbC homologs showed that the key active-site residues are conserved across kingdoms.
Topics: Bacillus anthracis; Bacterial Proteins; Carbohydrate Epimerases; Catalytic Domain; Crystallography, X-Ray; Diphosphates; Models, Molecular; Protein Conformation; Structural Homology, Protein
PubMed: 29199987
DOI: 10.1107/S2053230X17015849 -
PloS One 2017Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and...
Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders.
Topics: Animals; Diphosphates; Disease Models, Animal; Female; Humans; Injections, Intraperitoneal; Kidney Calculi; Male; Mice; Mice, Knockout; Mutation; Phosphoric Diester Hydrolases; Pyrophosphatases; Sodium-Phosphate Cotransporter Proteins, Type IIa; Treatment Outcome
PubMed: 28704395
DOI: 10.1371/journal.pone.0180098 -
The Journal of Biological Chemistry Dec 2016Previous measurements of the rates of polymerization and pyrophosphate release with DNA templates showed that pyrophosphate (PP) dissociation was fast after nucleotide...
Previous measurements of the rates of polymerization and pyrophosphate release with DNA templates showed that pyrophosphate (PP) dissociation was fast after nucleotide incorporation so that it did not contribute to enzyme specificity (k/K). Here, kinetic parameters governing nucleotide incorporation and PP release were determined using an RNA template. Compared with a DNA template of the same sequence, the rate of chemistry increased by up to 10-fold (250 versus 24 s), whereas the rate of PP release decreased to approximately 58 s so that PP release became the rate-limiting step. During processive nucleotide incorporation, the first nucleotide (TTP) was incorporated at a fast rate (152 s), whereas the rates of incorporation of remaining nucleotides (CGTCG) were much slower with an average rate of 24 s, suggesting that sequential incorporation events were limited by the relatively slow PP release step. The accompanying paper shows that slow PP release allows polymerization and RNase H to occur at comparable rates. Although PP release is the rate-determining step, it is not the specificity-determining step for correct incorporation based on our current estimates of the rate of reversal of the chemistry step (3 s). In contrast, during misincorporation, PP release became extremely slow, which we estimated to be ∼0.002 s These studies establish the mechanistic basis for DNA polymerase fidelity during reverse transcription and provide a free energy profile. We correct previous underestimates of discrimination by including the slow PP release step. Our current estimate of 2.4 × 10 is >20-fold greater than estimated previously.
Topics: Diphosphates; HIV Reverse Transcriptase; HIV-1; Ribonuclease H
PubMed: 27777304
DOI: 10.1074/jbc.M116.753152 -
The Journal of Biological Chemistry Jul 1984Studies on the interaction of calcineurin with its activator, calmodulin, showed that the 1:1 complex is the activated species. Concomitant with activation, a...
The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme.
Studies on the interaction of calcineurin with its activator, calmodulin, showed that the 1:1 complex is the activated species. Concomitant with activation, a time-dependent deactivation of the phosphatase was observed. The process followed first order kinetics and was dependent on the presence of both Ca2+ and calmodulin. The deactivation rate constant at pH 7.6 and 30 degrees C was 0.06 min-1, which was increased by the substrate, p-nitrophenylphosphate (Km = 11 mM), to 0.47 min-1. PPi and nucleotides inhibited the enzyme competitively and accelerated the deactivation. The first order rate constant was increased to 2.3 min-1 by PPi (Ki = 55 microM) and to 8.0 min-1 by ADP (Ki = 0.94 mM). A theory dealing with the deactivation (applicable to chemical modification, etc.) of an enzyme in the absence and presence of various ligands is presented. The deactivated enzyme remained bound to calmodulin and was not reactivated by dissociation-reassociation of the calcineurin-calmodulin complex. Calcineurin was found to contain covalently bound phosphate; however, no difference in its content was detected upon deactivation, indicating that self-dephosphorylation was not involved. The deactivation could be reversed, as well as prevented, by divalent metal ions such as Ni2+ and Mn2+. Atomic absorption spectroscopy revealed nearly stoichiometric amounts of tightly bound Fe and Zn (but little other ions) on purified calcineurin, which remained bound during the calmodulin-dependent deactivation; removal of tightly bound metals is, therefore, not the cause of deactivation. Our results indicate that calcineurin is a metallophosphatase and not simply a Ca2+- and calmodulin-stimulated enzyme. In addition to the nondissociable Zn and Fe and the Ca2+ bound to the B subunit and calmodulin, the enzyme requires a divalent metal ion for structural stability and full activity.
Topics: Adenosine Diphosphate; Animals; Brain; Calmodulin; Calmodulin-Binding Proteins; Cations, Divalent; Cattle; Diphosphates; Enzyme Activation; Iron; Kinetics; Mathematics; Models, Biological; Phosphoprotein Phosphatases; Zinc
PubMed: 6086614
DOI: No ID Found -
Organic & Biomolecular Chemistry Feb 2011P(1),P(2)-Diimidazolyl derivatives of pyrophosphate and halomethylene-bis-phosphonates have been synthesized and characterized, and the mechanism of their formation was...
P(1),P(2)-Diimidazolyl derivatives of pyrophosphate and halomethylene-bis-phosphonates have been synthesized and characterized, and the mechanism of their formation was studied. These reagents enable synthesis of dinucleoside tetraphosphates and tetraphosphonates conveniently and in high yields.
Topics: Cyclization; Dinucleoside Phosphates; Diphosphates; Imidazoles; Molecular Structure; Organophosphonates
PubMed: 21082127
DOI: 10.1039/c0ob00542h -
FEBS Letters Feb 2011Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on...
Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the "missing" GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.
Topics: Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins; Cell Wall; Dimethylallyltranstransferase; Diphosphates; Diterpenes; Farnesol; Flame Ionization; Gas Chromatography-Mass Spectrometry; Geranyltranstransferase; Hemiterpenes; Isomerism; Kinetics; Molecular Sequence Data; Mycobacterium tuberculosis; Organophosphorus Compounds; Polyisoprenyl Phosphates; Recombinant Proteins; Sequence Alignment; Sesquiterpenes; Substrate Specificity
PubMed: 21237161
DOI: 10.1016/j.febslet.2011.01.007