pyrophosphate and imidodiphosphonic-acid

Tissue-nonspecific alkaline phosphatase (TNAP) is known to be involved in the degradation of extracellular ATP via the hydrolysis of pyrophosphate (PPi). We investigated, using three different computational methods, namely molecular docking, thermodynamic integration (TI) and conventional molecular dynamics (MD), whether TNAP may also be involved in the utilization of β,γ-modified ATP analogues. For that, we analyzed the interaction of bisphosphonates with this enzyme and evaluated the obtained structures using in silico studies. Complexes formed between pyrophosphate, hypophosphate, imidodiphosphate, methylenediphosphonic acid monothiopyrophosphate, alendronate, pamidronate and zoledronate with TNAP were generated and analyzed based on ligand docking, molecular dynamics and thermodynamic integration. The obtained results indicate that all selected ligands show high affinity toward this enzyme. The forming complexes are stabilized through hydrogen bonds, electrostatic interactions and van der Waals forces. Short- and middle-term molecular dynamics simulations yielded very similar affinity results and confirmed the stability of the protein and its complexes. The results suggest that certain effectors may have a significant impact on the enzyme, changing its properties.

Topics: Adenosine Triphosphate; Alendronate; Alkaline Phosphatase; Computational Biology; Diphosphates; Diphosphonates; Enzymes; Humans; Hydrogen Bonding; Ligands; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Pamidronate; Phosphates; Protein Conformation; Thermodynamics; Zoledronic Acid

Imidodiphosphate (the pyrophosphate analog containing a nitrogen atom in the bridge position instead of oxygen) is a potent inhibitor of family II pyrophosphatases from Streptococcus mutans and Streptococcus gordonii (inhibition constant Ki approximately 10 microM), which is slowly hydrolyzed by these enzymes with a catalytic constant of approximately 1 min(-1). Diphosphonates with different substituents at the bridge carbon atom are much less effective (Ki = 1-6 mM). The value of Ki for sulfate (a phosphate analog) is only 12 mM. The inhibitory effect of the pyrophosphate analogs exhibits only a weak dependence on the nature of the metal ion (Mn, Mg, or Co) bound in the active site.

Topics: Cobalt; Diphosphates; Diphosphonates; Dose-Response Relationship, Drug; Hydrolysis; Kinetics; Magnesium; Manganese; Molecular Structure; Phosphates; Pyrophosphatases; Streptococcus; Streptococcus mutans; Substrate Specificity

The addition of exogenous pyrophosphate increases the growth yield and cAMP synthesis in stationary phase when Escherichia coli is grown in minimal medium. Pyrophosphate increases the yield by altering the enterobactin uptake system. We studied the physiological effects and examined how the E. coli transcriptome was modified when two structural analogs of pyrophosphate were added to the growth medium. Methylenediphosphonic acid or a high concentration of iron had the same positive effects as pyrophosphate on growth yield, cAMP synthesis and the repression of Fur-regulated genes. In contrast, imidodiphosphate did not affect these cellular processes significantly. The transcriptome modifications generated by pyrophosphate or methylenediphosphonic acid were more similar than those generated by imidodiphosphate or excess iron. The transcriptome data also indicated that processes other than iron uptake might be involved in the cellular response to exogenous pyrophosphate or methylenediphosphonic acid.

Topics: Culture Media; Cyclic AMP; Diphosphates; Diphosphonates; Escherichia coli; Escherichia coli Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Iron; Oligonucleotide Array Sequence Analysis; Proteome; Transcription, Genetic

We previously reported the isolation from bovine liver of a novel 56-kDa inorganic pyrophosphatase named phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase). It is a unique enzyme that hydrolyzes not only oxygen-phosphorus bonds in inorganic pyrophosphate but also nitrogen-phosphorus bonds in phospholysine, phosphohistidine and imidodiphosphate in vitro. In this study, we determined the partial amino acid sequence of the purified bovine LHPPase. To investigate whether humans have the same enzyme, we isolated a cDNA clone from a HeLa cell cDNA library that encodes for the human homologue of LHPPase. Although its sequence does not include the consensus sequence of a typical inorganic pyrophosphatase, it does contain a similar sequence of the active site in other phosphatases such as protein-tyrosine phosphatase, dual-specific phosphatase and low molecular weight acid phosphatase. Human LHPPase was highly expressed in the liver and kidney, and moderately in the brain. The recombinant protein was produced in E. coli. Its ability to hydrolyze oxygen-phosphorus bonds and nitrogen-phosphorus bonds was confirmed. The enzymatic characteristics of this human protein were similar to those of purified bovine LHPPase. Thus, we concluded that the cDNA encoded the human counterpart of bovine LHPPase.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cattle; Cloning, Molecular; Diphosphates; Diphosphonates; DNA, Complementary; Enzyme Inhibitors; Fetus; Humans; Hydrogen-Ion Concentration; Inorganic Pyrophosphatase; Molecular Sequence Data; Molecular Weight; Organomercury Compounds; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Tissue Distribution

S-Adenosylmethionine (AdoMet) synthetase catalyzes the only known route of biosynthesis of the primary in vivo alkylating agent. Inhibitors of this enzyme could provide useful modifiers of biological methylation and polyamine biosynthetic processes. The AdoMet synthetase catalyzed reaction converts ATP and L-methionine to AdoMet, PP(i), and P(i), with formation of tripolyphosphate as a tightly bound intermediate. This work describes a nonhydrolyzable analogue of the tripolyphosphate (PPP(i)) reaction intermediate, diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3)(5)(-)), as a potent inhibitor. In the presence of AdoMet, PNPNP is a slow-binding inhibitor with an overall inhibition constant (K(i)) of 2 nM and a dissociation rate of 0.6 h(-)(1). In contrast, in the absence of AdoMet PNPNP is a classical competitive inhibitor with a K(i) of 0.5 microM, a slightly higher affinity than PPP(i) itself (K(i) = 3 microM). The imido analogue of the product pyrophosphate, imidodiphosphate (O(3)P-NH-PO(3)(4)(-)) also displays slow onset inhibition only in the presence of AdoMet, with a K(i) of 0.8 microM, compared to K(i) of 250 microM for PP(i). Circular dichroism spectra of the unliganded enzyme and various complexes are indistinguishable indicating that the protein secondary structure is not greatly altered upon complex formation, suggesting local rearrangements at the active site during the slow binding process. A model based on ionization of the bridging -NH- moiety is presented which could account for the potent inhibition by PNP and PNPNP.

Topics: Acid Anhydride Hydrolases; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Amino Acid Substitution; Arginine; Binding, Competitive; Diphosphates; Diphosphonates; Enzyme Inhibitors; Hydrolysis; Leucine; Methionine Adenosyltransferase; Mutagenesis, Site-Directed; Polyphosphates

The structure of sodium imidodiphosphate has been determined by single crystal x-ray diffraction. The P-N-P bond angle (127.2 degrees) and P-N bond distance (1.68 angstroms) are remarkably similar to newly refined values for the P-O-P bond angle (128.6 degrees) and the bridging P-O bond distance (1.63 angstroms) of sodium pyrophosphate. This close similarity may explain why P-N-P linkages in algal "polyphosphates" escaped detection until recently and why adenosine triphosphate analogs with this linkage mimic adenosine triphosphate so closely.

Topics: Adenosine Triphosphate; Chemical Phenomena; Chemistry, Physical; Crystallography, X-Ray; Diphosphates; Diphosphonates; Models, Chemical