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PubMed1997

Phosphatidate phosphatases and diacylglycerol pyrophosphate phosphatases in Saccharomyces cerevisiae and Escherichia coli.

Phosphatidate phosphatase plays a major role in the synthesis of phospholipids and triacylglycerols in the yeast Saccharomyces cerevisiae. Membrane- and cytosolic-associated forms of the enzyme have been isolated and characterized. These enzymes are Mg2+-dependent and N-ethylmaleimide-sensitive. The expression of a membrane-associated form of phosphatidate phosphatase is regulated by growth phase and inositol supplementation, whereas enzyme activity is regulated by lipids, nucleotides, and by phosphorylation. Phosphatidate phosphatase is coordinately regulated with other phospholipid biosynthetic enzymes including phosphatidylserine synthase. Diacylglycerol pyrophosphate phosphatase is a novel enzyme of phospholipid metabolism which is present in S. cerevisiae, Escherichia coli, and mammalian cells. This enzyme possesses a phosphatidate phosphatase activity which is Mg2+-independent and N-ethylmaleimide-insensitive and is distinct from the Mg2+-dependent and N-ethylmaleimide-sensitive form of phosphatidate phosphatase. Genes encoding for diacylglycerol pyrophosphate phosphatase have been isolated from S. cerevisiae and E. coli. The deduced protein sequences of these genes show homology to the sequence of the mouse PAP2 (Mg2+-independent and N-ethylmaleimide-insensitive phosphatidate phosphatase) protein, especially in a novel phosphatase sequence motif. Rat liver PAP2 displays diacylglycerol pyrophosphate phosphatase activity.

Amino Acid Sequence↗

PubMed1986

Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose importing plant tissues.

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.

Biological Transport↗

PubMed1997

A search for pyrophosphate mimics for the development of substrates and inhibitors of glycosyltransferases.

The design and synthesis of several beta-1,4-galactosyltransferase inhibitors are reported. Mimics of the pyrophosphate-Mn2+ complex were the focus of the design. Malonic, tartaric, and monosaccharide moieties were used as replacements of the pyrophosphate moiety, and galactose or azasugars with potent galactosidase inhibitory activity were used as the 'donor' component. Compound 6, in which glucose was used as the pyrophosphate-Mn2+ complex mimic and galactose as the 'donor' component, showed the best inhibitory activity towards the transferase with a Ki of 119.6 microM.

Aminoglycosides↗

PubMed1995

X-ray structure of the magnesium(II)-pyrophosphate complex of the truncated head of Dictyostelium discoideum myosin to 2.7 A resolution.

The structure of the magnesium pyrophosphate complex of the truncated head of Dictyostelium myosin has been determined by molecular replacement at 2.7 A resolution and refined to a crystallographic R-factor of 16.0%. The crystals belong to the orthorhombic space group P2(1)2(1)2, where a = 105.2 A, b = 182.1 A, and c = 54.5 A. The conformation of the protein around the magnesium pyrophosphate is very similar to that seen when magnesium ADP-beryllium fluoride binds in the active site. The latter complex mimics the binding of ATP prior to hydrolysis. The pyrophosphate molecule occupies the beta- and gamma-phosphate sites, where the two phosphorus atoms are in the same positions as the beta-phosphate and the BeFx moiety of the beryllium fluoride-trapped ADP. The surrounding active site residues are almost perfectly superimposable in the two structures and the hydrogen-bonding interactions that the PPi makes with the protein are essentially identical. The similarity between the MgPPi and MgADP.BeFx complex with S1Dc suggests that the conformational change, which occurs when ATP binds to actomyosin and which reduces the affinity of myosin for actin, is caused by the binding of the gamma- and beta-phosphate groups of the nucleotide. This then implies that the role of the remainder of the substrate is to increase the binding affinity for myosin and thus to drive the equilibrium toward dissociation of myosin from actin.

Adenosine Diphosphate↗

PubMed1992

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.

Amino Acid Sequence↗

PubMed1985

Contribution of water to free energy of hydrolysis of pyrophosphate.

The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes. With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol. The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C. All the cosolvents used promoted a decrease of Kobsd. Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd. The higher the molecular weight of the polymer, the lower the value of Kobsd. A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0. The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate. A decrease in all the Keq was observed. The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products.

Diphosphates↗

PubMed1988

Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase.

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.

Carbon Radioisotopes↗

PubMed1980

Effects of pyrophosphate, triphosphate, and potassium chloride on adenylate deaminase from rat muscle.

Inorganic pyrophosphate and triphosphate inhibit adenylate deaminase from rat skeletal muscle with K1 values of 10 and 1.5 microM, respectively, in the presence of 150 mM KCl at pH 7. They act by reducing the apparent affinity of the enzyme for AMP, with relatively small effects on Vmax. The inhibitions are diminished by H+, the KI values increasing two- to threefold in going from pH 7.0 to 6.2, and are relieved by ADP. These properties are similar to the inhibitions produced by GTP and ATP, indicating that pyrophosphate and triphosphate act like analogues of the nucleoside triphosphates. Neither of these inhibitors shows relief of inhibition at high concentrations as do ATP and GTP. These results suggest that nucleotides interact with the inhibitor site of the enzyme primarily through their phosphate moieties and with the activator site primarily through their nucleoside moieties. As the concentration of KCl is increased from 25 to 300 mM, the apparent affinities of the enzyme for ATP, GTP, orthophosphate, pyrophosphate, and triphosphate are decreased 8-100-fold. The cooperativity of the inhibitions is increased with the Hill coefficient rising from 1.0 to 1.3-1.8, and the maximum inhibition approaches 100%. Maximum activation by ADP is reduced from 1800% at 25 mM KCl to 80% at 200 mM KCl. Experiments with (CH3)4NCl indicate that activation of the enzyme by KCl involves both specific K+ effects and ionic strength effects.

AMP Deaminase↗

PubMed1978

Purification and characterization of dimethylallyl pyrophosphate: aspulvinone dimethylallyltransferase from Aspergillus terreus.

Dimethylallyl pyrophosphate:aspulvinone dimethylallyltransferase, the prenylation enzyme for the biosynthesis of aspulvinone pigments, has been purified from mycelia of Aspergillus terreus. The transferase catalyzed the transfer of the dimethylallyl moiety from dimethylallyl pyrophosphate to either of the two aromatic rings of aspulvinone E to give the mono- and diprenylated derivatives which were identified with the metabolites aspulvinone I and aspulvinone H, respectively. Aspulvinone G, another fundamental metabolite of this series, also acted as substrate to afford the corresponding diprenylated derivative, which is assumed to be a precursor for aspulvinone C, D, and F. The molecular weight of the enzyme was estimated to be 240 000--270 000 by gel filtration. Since the subunit molecular weight determined by NaDodSO4-polyacrylamide disc gel electrophoresis was 45 000, the native enzyme appears to be a hexomeric protein composed of identical molecular weight subunits. The apparent Km values for aspulvinone E, aspulvinone G, and dimethylallyl pyrophosphate were 13.7, 7.7, and 40.0 micron, respectively. The enzyme shows the maximum activity at pH 7.0, and no metal ion is necessary for the activation. Sulfhydryl blocking agents or mercaptoethanol has no effect. Bromophenol blue binds specifically to the transferase and strongly inhibits the enzyme activity.

Alkyl and Aryl Transferases↗

PubMed1999

Determination by Multi-NMR Studies of New Stable Aluminum Complexes with Pyrophosphate and Fluoride in Aqueous Solution.

Multi-NMR studies ((27)Al, (19)F, (31)P) show that, in the presence of pyrophosphate at pH 7, fluoride and aluminum (Al(3+)) ions combine in aqueous solution, to generate a wide variety of ternary complexes. Among the latter, a new class of stable complexes, showing J(FF) and J(FP) coupling constants, has been identified in which two octahedral aluminum cations are bound by two pyrophosphate moieties, with a common fluorine atom bridging the two aluminum ions. Both the pyrophosphate groups and the fluoride ion contribute to the stability of these molecules. The existence of J(FF) couplings provides evidence for the octahedral coordination of aluminum in these complexes.

Journal Article↗

PubMed2005

A crystallographic investigation of phosphoantigen binding to isopentenyl pyrophosphate/dimethylallyl pyrophosphate isomerase.

We report the crystallographic structures of the potent phosphoantigens Phosphostim (the bromohydrin of isopentenyl pyrophosphate) and E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate bound to the mevalonate pathway enzyme isopentenyl pyrophosphate/dimethylallyl pyrophosphate isomerase (IPPI). Racemic Phosphostim forms covalent complexes with IPPI: a 4-thioether with C67 and a 4-ester with E116. Only the E116 ester forms with the chiral species, S-Phosphostim, with the w.t. enzyme, while the C67 thioether forms with a mutant Y104F IPPI. The potent phosphoantigen HMBPP also binds to IPPI, but is only a weak ( approximately 50 muM) inhibitor. These results strongly support an SN2 reaction for inhibition of IPPI by Phosphostim, in contrast to the SN1 or concerted type of reaction found with epoxide inhibitors, which react at C-3, and are of general interest in the context of the development of novel mevalonate pathway inhibitors. They also provide clues as to the nature of the binding site of synthetic phosphoantigens in gammadelta T cell activation. In particular, both bromohydrin and epoxy phosphoantigens are potent, irreversible inhibitors of IPPI while HMBPP is only a weak inhibitor, ruling out an IPPI or IPPI-like target for HMBPP in gammadelta T cell activation.

Antigens↗

PubMed2004

Improvement of functional properties of egg white protein through phosphorylation by dry-heating in the presence of pyrophosphate.

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.

Diphosphates↗

PubMed2000

An efficient synthesis of cyclic IDP- and cyclic 8-bromo-IDP-carbocyclic-riboses using a modified Hata condensation method to form an intramolecular pyrophosphate linkage as a key step. An entry to a general method for the chemical synthesis of cyclic ADP-ribose analogues

An efficient synthesis of cyclic IDP-carbocyclic-ribose (3) and its 8-bromo derivative 6, as stable mimics of cyclic ADP-ribose, was achieved, and a condensation reaction with phenylthiophosphate-type substrate 15 or 16 to form an intramolecular pyrophosphate linkage was a key step. N-1-Carbocyclic-ribosylinosine derivative 28 and the corresponding 8-bromo congener 24 were prepared via condensation between N-1-(2,4-dinitrophenyl)inosine derivative 17 and a known optically active carbocyclic amine 18. Compounds 24 and 28 were then converted to the corresponding 5"-phosphoryl-5'-phenylthiophosphate derivatives 15 and 16, respectively, which were substrates for the condensation reaction to form an intramolecular pyrophosphate linkage. Treatment of 8-bromo substrate 15 with I2 or AgNO3 in the presence of molecular sieves 3A (MS 3A) in pyridine at room temperature gave the desired cyclic product 12 quantitatively, while the yield was quite low without MS. The similar reaction of 8-unsubstituted substrate 16 gave the corresponding cyclized product 32 in 81% yield. Acidic treatment of these cyclic pyrophosphates 12 and 32 readily gave the targets 6 and 3, respectively. This result suggests that the construction of N-1-substituted hypoxanthine nucleoside structures from N-1-(2,4-dinitrophenyl)inosine derivatives and the intramolecular condensation by activation of the phenylthiophosphate group with I2 or AgNO3/MS 3A combine to provide a very efficient route for the synthesis of analogues of cyclic ADP-ribose such as 3 and 6. Thus, this may be an entry to a general method for synthesizing biologically important cyclic nucleotides of this type.

Journal Article↗

PubMed2005

Highly effective fluorescent and colorimetric sensors for pyrophosphate over H2PO4- in 100% aqueous solution.

[Structure: see text]. This study demonstrated that Zinpyr-1*Zn2+ acts as a fluorescent and colorimetric sensor for pyrophosphate at pH 7.4. In addition, Zinpyr-1*Cu2+ and DIARB-1*Cu2+ complexes were found to act as selective fluorescent sensors for pyrophosphate. Furthermore, the chemosensors Zinpyr-1*Zn2+ and Zinpyr-1*Cu2+ show highly selective and ratiometric fluorescence changes for pyrophosphate compared with H2PO4-.

Biosensing Techniques↗

PubMed2002

Rat osseous plate alkaline phosphatase: effect of neutral protease digestion on the hydrolysis of pyrophosphate and nitrophenylphosphate.

Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1,000 U/mL collagenase for 3 h, at 37 degrees C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates. The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence ofcollagenase treatment. In the absence of Mg2+ ions, the enzyme hydrolyzed PNPP with KM = 322.5 +/- 15.3 microM and V = 965.2 +/- 45.8 U/mg, while in the presence of 2 mM Mg2+ ions, V increased 66%. Cobalt (K0.5 = 5.3 +/- 0.3 microM) and manganese (K0.5 = 0.72 +/- 0.03 microM) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg2+ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (KM = 105.1 +/- 6.3 microM and V = 64.9 +/- 3.9 U/mg), but site-site interactions (nH = 1.2) were observed in the presence of 2 mM Mg2+ ions (V = 110.8 +/- 5.5 U/mg; K0.5 = 42.7 +/- 2.0 microM). To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.

Alkaline Phosphatase↗

PubMed1973

Synthesis, characterization and properties of uridine 5'-( -D-apio-D-furanosyl pyrophosphate).

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.

Carbon Isotopes↗

PubMed1979

Inhibition of rat liver mevalonate pyrophosphate decarboxylase and mevalonate phosphate kinase by phenyl and phenolic compounds.

1. Mevalonate pyrophosphate decarboxylase of rat liver is inhibited by various phenyl and phenolic acids. 2. Some of the phenyl and phenolic acids also inhibited mevalonate phosphate kinase. 3. Compounds with the phenyl-vinyl structure were more effective. 4. Kinetic studies showed that some of the phenolic acids compete with the substrates, mevalonate 5-phosphate and mevalonate 5-pyrophosphate, whereas others inhibit umcompetitively. 5. Dihydroxyphenyl and trihydroxyphenyl compounds and p-chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. 6. Of the three mevalonate-metabolizing enzymes, mevalonate pyrophosphate decarboxylase has the lowest specific activity and is probably the rate-determining step in this part of the pathway.

Animals↗

PubMed2003

Calcium pyrophosphate dihydrate crystal deposition disease.

Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is characterized by the accumulation of pyrophosphate dihydrate crystals in articular and periarticular tissues. Various terms have been utilized to describe this arthropathy, which has led to some confusion. CPPD crystal deposition disease is among many conditions that may result in crystal deposition within cartilage. Chondrocalcinosis is a pathologic and radiographic term denoting calcification of cartilage within joints including both hyaline articular cartilage and fibrocartilage. Pseudogout is a clinical term applied to an acute inflammatory process in a joint(s) mimicking a gout attack. Pseudogout is just one of the multiple clinical presentations for CPPD crystal deposition disease. Pyrophosphate arthropathy is a term that has been used to describe the peculiar pattern of joint destruction associated with CPPD crystal deposition disease. This article reviews the protean manifestations of CPPD crystal deposition disease with emphasis on diagnostic imaging.

Arthrography↗