pyrophosphate and monothiopyrophosphoric-acid

Differential scanning calorimetry (DSC) and low-temperature X-ray diffraction studies showed that 2-thio-(5,5-dimethyl-1,3,2-dioxaphosphorinanyl)2'-oxo-dineopentyl-thiophosphate (compound 1) undergoes reversible phase transition at 203 K related to the change of symmetry of the crystallographic unit. Solid state NMR spectroscopy was used to establish the dynamic processes of aliphatic groups and the phosphorus skeleton. 13C and 31P variable temperature NMR studies as well as T1 and T1rho measurements of relaxation times revealed the different mode of molecular motion for each neopentyl residue directly bonded to phosphorus. It is concluded that molecular dynamics of aliphatic groups causes different van der Waals interactions in the crystal lattice and is the driving force of phase transition for compound 1. Finally, we showed that very sharp phase transition temperature makes compound 1 an excellent candidate as a low-temperature NMR thermometer in the solid phase.

Topics: Diphosphates; Magnetic Resonance Spectroscopy; Models, Molecular; X-Ray Diffraction

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi

Topics: Anti-HIV Agents; Binding Sites; Combinatorial Chemistry Techniques; Diphosphates; Electrophoretic Mobility Shift Assay; Gene Library; HIV Reverse Transcriptase; HIV-1; Magnetic Resonance Spectroscopy; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Structure, Tertiary; Reverse Transcriptase Inhibitors; Ribonuclease H; Thionucleotides; Virus Replication

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate (UPP). In the present study, site-directed mutagenesis, fluorescence quenching, and stopped-flow methods were utilized to examine the substrate binding and the protein conformational change. (S)-Farnesyl thiopyrophosphate (FsPP), a FPP analogue, was synthesized to probe the enzyme inhibition and events associated with the protein fluorescence change. This compound with a much less labile thiopyrophosphate shows K(i) value of 0.2 microm in the inhibition of Escherichia coli UPPS and serves as a poor substrate, with the k(cat) value (3.1 x 10(-7) s(-1)) 10(7) times smaller than using FPP as the substrate. Reduction of protein intrinsic fluorescence was observed upon addition of FPP (or FsPP) to the UPPS solution. Moreover, fluorescence studies carried out using W91F and other mutant UPPS with Trp replaced by Phe indicate that FPP binding mainly quenches the fluorescence of Trp-91, a residue in the alpha3 helix that moves toward the active site during substrate binding. Using stopped-flow apparatus, a three-phase protein fluorescence change with time was observed by mixing the E.FPP complex with IPP in the presence of Mg(2+). However, during the binding of E.FsPP with IPP, only the fastest phase was observed. These results suggest that the first phase is due to the IPP binding to E.FPP complex, and the other two slow phases are originated from the protein conformational change. The two slow phases coincide with the time course of FPP chain elongation from C(15) to C(55) and product release.

Topics: Alkyl and Aryl Transferases; Binding Sites; Carbon; Catalysis; Chromatography, High Pressure Liquid; Diphosphates; Dose-Response Relationship, Drug; Escherichia coli; Inhibitory Concentration 50; Kinetics; Magnetic Resonance Spectroscopy; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Phenylalanine; Protein Binding; Protein Conformation; Spectrometry, Fluorescence; Time Factors; Tryptophan

The tris(tetra-n-butylammonium) salt of thiopyrophosphate 5 was prepared from trimethyl phosphate in four steps. Treatment of geranyl bromide with 5 gave an 80% yield of geranyl S-thiolodiphosphate (6). Thiolodiphosphate 6 is substantially less reactive than geranyl diphosphate (7) in the prenyl transfer reaction catalyzed by farnesyl diphosphate synthase and is a good inhibitor of the enzyme.

Topics: Alkyl and Aryl Transferases; Dimethylallyltranstransferase; Diphosphates; Enzyme Inhibitors; Geranyltranstransferase; Organothiophosphorus Compounds

mu-Monothiopyrophosphate (MTP, (2-)O3P-S-PO3(-2)) is an excellent substrate for inorganic pyrophosphatase. The maximum velocity for the hydrolysis of MgMTP by inorganic pyrophosphatase is 24% of that for MgPPi at pH 8.0 and 5 degrees C and 190% at pH 9.0 and 15 degrees C. The hydrolyses of MnMTP and CoMTP proceed at 24 and > or = 7%, respectively, of the maximum velocity for the reaction of MgMTP at pH 8 and 5 degrees C. The maximum velocities for hydrolyses of MnPPi and CoPPi are 31 and 13% of that for MgPPi, respectively. There is no evidence that Mn2+ or Co2+ coordinate with bridging sulfur in MTP in such a way as to affect the rate of hydrolysis. The apparent Michaelis constants at pH 8 and 5 degrees C in the presence of 195 microM divalent metal ion are as follows: MgPPi, 12 microM; MnPPi, 19 microM; CoPPi, 12 microM; MgMTP, 45 microM; MnMTP, 5.3 microM; and CoMTP, 16 microM. The apparent Michaelis constants at pH 9 and 15 degrees C in the presence of 10 mM divalent metal ion are MgPPi, 1.9 microM and MgMTP, 19.1 microM. The values of kcat for MgPPi at pH 8 and 5 degrees C and at pH 9 and 15 degrees C are 8 s(-1) and 12.4 s(-1), respectively. The values of kcat for MgMTP under the same conditions are 2 s(-1) and 24 s(-1), respectively. MTP and MgMTP undergo nonenzymatic hydrolysis by a mechanism in which monomeric metaphosphate monoanion (PO3) is a discrete intermediate (Lightcap, E.S., and Frey, P.A. (1992) J. Am. Chem. Soc. 114, 9750-9755). This reaction is accommodated at the active site of inorganic pyrophosphatase, indicating that the mechanism of enzymatic hydrolysis is dissociative. MgMTP is also a substrate for UDP-glucose pyrophosphorylase, reacting at 4.8% of the maximum velocity of MgPPi and with a Michaelis constant 17 times larger than that for MgPPi. The P-S bonds of MgMTP are not cleaved in the pyrophosphorylase reaction, but the product UTP beta gamma S is chemically unstable and undergoes hydrolysis to UDP beta S and Pi, making the cleavage of UDP-glucose to glucose-1-P, UDP beta S and Pi, experimentally irreversible.

Topics: Antiviral Agents; Cations, Divalent; Cobalt; Diphosphates; Inorganic Pyrophosphatase; Kinetics; Manganese; Models, Chemical; Pyrophosphatases; Saccharomyces cerevisiae; Substrate Specificity; UTP-Glucose-1-Phosphate Uridylyltransferase

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cations, Divalent; Cations, Monovalent; Diphosphates; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Hydrolysis; Metals; Structure-Activity Relationship; Thermodynamics

mu-Monothiopyrophosphate (MTP), an analogue of pyrophosphate (PPi) with sulfur in place of oxygen in the bridge position, is a substrate for the enzyme pyrophosphate-dependent phosphofructokinase. At pH 9.4 and 6 degrees C, the maximal velocity for the phosphorylation of fructose 6-phosphate (F6P) by MgMTP is about 2.8% of that with MgPPi as the phosphoryl donor. The kinetic mechanism is equilibrium random with rate-limiting transformation of the substrate ternary complex to the product when either MgMTP or MgPPi is the phosphoryl donor. This is known from independent studies to be kinetic mechanism at pH 8.0 and 25 degrees C [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101-4108]. The dissociation constant of MgPPi is 14 microM, that of MgMTP is 64 microM, and that of F6P from the enzyme is about 5 mM. The Km values for MgPPi and MgMTP are 14.5 and 173 microM, respectively. MgMTP competes with MgPPi for binding to the enzyme. The values of kcat are 3.4 s-1 and 140 s-1 for MgMTP and MgPPi, respectively, at pH 9.4 and 6 degrees C. The estimated rate enhancement factors are 3.6 x 10(5) and 1.4 x 10(14) for the reactions of MgMTP and MgPPi, respectively. Therefore, MgMTP is a reasonably good substrate for PPi-dependent PKF, on the basis of comparisons of kcat. However, the rate enhancement factors show that the enzyme is a poor catalyst for the reaction of MgMTP. Lesser enzymatic catalysis in the reaction of MgMTP compared with MgPPi is largely compensated for by the greater intrinsic reactivity of MgMTP. Thus, the larger substrate MgMTP is well accommodated in the active site, and the dissociative reaction of MgMTP is well accommodated in the transition state. The results are interpreted to indicate a dissociative transition state for phosphoryl group transfer by PPi-dependent PFK. A modified synthesis and purification of MTP are described, in which (trimethylsilyl)trifluoromethanesulfonate and tetra-N-butylammonium iodide are used in place of iodotrimethylsilane to dealkylate tetramethyl-MTP.

Topics: Animals; Binding, Competitive; Diphosphates; Fructosephosphates; Kinetics; Phosphorylation; Phosphotransferases; Rabbits; Substrate Specificity

31P-nuclear magnetic resonance spectroscopy was used to directly determine the equilibrium of the reaction catalysed by yeast orotate phosphoribosyltransferase, using orotidine monophosphate and inorganic pyrophosphate as substrates. A Keq value of 0.71 was determined, in good agreement with that of 0.49 calculated by Victor, Greenberg and Sloan (J. Biol. Chem. 254 (1979) 2647-2655), from kinetic data. Substitution of thiopyrophosphate as the substrate shifted the position of the equilibrium 55-fold, to yield a Keq value of 39. Only the beta S analogue of 5-phosphoribosyl 1-diphosphate appeared to be synthesized in this reaction.

Topics: Diphosphates; Magnetic Resonance Spectroscopy; Orotate Phosphoribosyltransferase; Pentosyltransferases; Saccharomyces cerevisiae; Thermodynamics

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.

Topics: Chromium; Cobalt; Diphosphates; Inorganic Pyrophosphatase; Kinetics; Magnesium; Phosphorus Radioisotopes; Protein Binding; Pyrophosphatases; Saccharomyces cerevisiae

Mono- and bisthiopyrophosphate can inhibit the replication of influenza virus A/X49 in Madin-Darby canine kidney (MDCK) cells at concentrations at which no cytotoxic effect is observed after 3 days. The thiopyrophosphate analogues inhibit the RNA transcriptase activity of this virus possibly by chelating with an essential metal ion in the transcriptase complex. [31P]NMR spectroscopy indicates that bisthiopyrophosphate coordinates to zinc through sulphur and magnesium through oxygen which may influence the inhibitory properties of this compound with metal-containing enzymes.

Topics: Antiviral Agents; Diphosphates; DNA-Directed RNA Polymerases; Influenza A virus; Virus Replication