24937-83-5

Basic information

• Product Name: POLYADENYLIC ACID POTASSIUM SALT
• Synonyms: polyadenylic acid potassium salt mr ~7000000;PolyadenylicAcid,M.W.>100,000
• CAS NO: 24937-83-5
• Molecular Formula: C10H14N5O7P
• Molecular Weight: 347.22
• Mol File: 24937-83-5.mol
• Article Data: 151

Chemical Properties

• Appearance: /
• Storage Temp.: −20°C
• CAS DataBase Reference: POLYADENYLIC ACID POTASSIUM SALT(CAS DataBase Reference)
• NIST Chemistry Reference: POLYADENYLIC ACID POTASSIUM SALT(24937-83-5)
• EPA Substance Registry System: POLYADENYLIC ACID POTASSIUM SALT(24937-83-5)

Safety Data

• WGK Germany: 3
• F: 3-10-21
• Hazardous Substances Data: 24937-83-5(Hazardous Substances Data)

Usage

General Description

Polyadenylic Acid Potassium Salt is a chemical compound used mainly in biotechnological applications such as in molecular biology, biochemistry, and genetic engineering. It is very vital in biological phenomena because it assists in processes such as RNA synthesis and translation, immunological activation, and molecular cloning. The compound comes in the form of a white to off-white crystalline powder that is easily soluble in water. It is denoted by the CAS number 30811-80-4 and has a molecular weight of 347.4 g/mol. Despite its numerous applications, it must be handled with care due to its hazardous effects.

InChI:InChI=1/C10H14N5O7P/c11-8-5-9(13-2-12-8)15(3-14-5)10-7(17)6(16)4(22-10)1-21-23(18,19)20/h2-4,6-7,10,16-17H,1H2,(H2,11,12,13)(H2,18,19,20)/t4-,6-,7-,10-/m1/s1

Upstream product

• 60-92-4 cAMP 
• 20408-44-0 2',3'-Isopropylidene-5'-AMP 
• 606-67-7 disodium adenosine-5' triphosphate 
• 58-64-0 adenosine 5'-diphosphate 
• 58-61-7 adenosine 
• 10025-87-3 trichlorophosphate 
• 917-08-8 diphosphoric acid-1-adenosin-5'-yl ester-2-[(1R)-1-(5-amino-4-carbamoyl-imidazol-1-yl)-D-1,4-anhydro-ribitol-5-yl ester] 
• 7732-18-5 water 
• 31528-64-0 [5']adenylic acid L-tryptophan anhydride 
• 79-14-1 glycolic Acid 
• 20816-58-4 adenosine 5'-phosphorimidazolide 
• 503-66-2 3-hydroxypropionic acid 
• 849585-22-4 LACTIC ACID 
• 591-81-1 4-hydroxybutanoic acid 

Downstream Products

• 13039-55-9 [5']adenylic acid monobenzyl ester 
• 13039-54-8 Adenosin-5'-phosphorsaeure-methylester 
• 121969-80-0 [5']adenylic acid-(N-benzyloxycarbonyl-L-isoleucine)-anhydride 
• 52435-67-3 Ser-AMP 
• 132129-32-9 [5']adenylic acid hexanoic acid-anhydride 
• 35985-26-3 glycyl adenylate 
• 117776-66-6 [5']adenylic acid-[N-(N-benzyloxycarbonyl-glycyl)-L-tyrosine]-anhydride 
• 13015-87-7 adenosine-5'-phosphoric acetic anhydride 
• 56164-09-1 [5']adenylic benzoic anhydride 
• 35908-06-6 [5']adenylic acid-(N-benzyloxycarbonyl-glycine)-anhydride 
• 19046-78-7 N-(purine-6-yl)-L-aspartic acid 9-β-D-ribofuranoside-5'-phosphate 
• 31528-64-0 [5']adenylic acid L-tryptophan anhydride 
• 6154-31-0 AMP-Amidate 
• 4061-78-3 Adenosine N'-oxide 5'-monophosphate 

Relevant articles and documents

Characterization of acetyl-CoA synthetase kinetics and ATP-binding

Background: The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. Methods: In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. Results: The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. Conclusions: The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General Significance: The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.

Synthesis and enhanced DNA cleavage activities of bis-tacnorthoamide derivatives

A new metal-free DNA cleaving reagent, bis-tacnorthoamide derivative 1 with two tacnorthoamide (tacnoa) units linked by a spacer containing anthraquinone, has been synthesized from triazatricyclo[5.2.1.04,10]decane and characterized by NMR and mass spectrometry. For comparison, the corresponding compounds mono-tacnorthoamide derivative 2 with one tacnorthoamide unit and 6 with two tacnorthoamide units linked by an alkyl (1,6-hexamethylene) spacer without anthraquinone have also been synthesized. The DNA-binding property investigated via fluorescence and CD spectroscopy suggests that compounds 1 and 2 have an intercalating DNA binding mode, and the apparent binding constants of 1, 2 and 6 are 1.3 × 107 M-1, 0.8 × 10 7 M-1 and 8 × 105 M-1, respectively. Agarose gel electrophoresis was used to assess plasmid pUC19 DNA cleavage activity promoted by 1, 2, 6 and parent tacnoa under physiological conditions, which gives rate constants kobs of 0.2126 ± 0.0055 h-1, 0.0620 ± 0.0024 h-1, 0.040 ± 0.0007 h-1 and 0.0043 ± 0.0002 h-1, respectively. The 50-fold and 15-fold rate acceleration over parent tacnoa is because of the anthraquinone moiety of compound 1 or 2 intercalating into DNA base pairs via a stacking interaction. Moreover, DNA cleavage reactions promoted by compound 1 give 5.3-fold rate acceleration over compound 6, which further demonstrates that the introduction of anthraquinone results in a large enhancement of DNA cleavage activity. In particular, DNA cleavage activity promoted by 1 bearing two tacnoa units is 3.3 times more effective than 2 bearing one tacnoa unit and the DNA cleavage by compound 1 was achieved effectively at a relatively low concentration (0.03 mM). This dramatic rate acceleration suggests the cooperative catalysis of the two positively charged tacnoa units in compound 1. The radical scavenger inhibition study and ESI-MS analysis of bis(2,4-dinitrophenyl) phosphate (BDNPP) and adenylyl(3′-5′) phosphoadenine (APA) cleavage in the presence of compound 1 suggest the cleavage mechanism would be via a hydrolysis pathway by cleaving the phosphodiester bond of DNA.

Thiamin biosynthesis in eukaryotes: Characterization of the enzyme-bound product of thiazole synthase from Saccharomyces cerevisiae and its implications in thiazole biosynthesis

The biosynthesis of thiamin pyrophosphate in eukaryotes is different from the prokaryotic biosynthesis and is poorly understood to date. Only one thiazole biosynthetic gene has been identified (Thi4 in Saccharomyces cerevisiae). Here we report the identification and characterization of a Thi4-bound metabolite that consists of the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid. The unexpected structure of this compound yields the first insights into the mechanism of thiamin thiazole biosynthesis in eukaryotes. Copyright

Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5′-O-sulfamoyl-N 6-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PPi exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.

Enzymatic synthesis of new pyridine nucleosides. Clitidine and its amide derivative

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Molecular characterization and mutational analysis of recombinant diadenosine 5′,5″-P1,P4-tetraphosphate hydrolase from Plasmodium falciparum

Asymmetrical diadenosine 5′,5″-P1,P 4-tetraphosphate hydrolase (EC 3.6.1.17) from human malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity, and characterized for the first time as a biological target for chemotherapeutic agents against malaria. Plasmodium falciparum Ap4A (PfAp4A) hydrolase not only catalyzes diadenosine 5′,5″-P1,P4-tetraphosphate (Ap4A) to ATP and AMP, but also diadenosine 5′,5″-P1,P 5-pentaphosphate (Ap5A) to ATP and ADP. Marked enzyme heat stability corresponding to the highest level of activity was observed at 60°C. The recombinant enzyme showed maximal activity in the presence of 5 mM Mg2+ ions. Kinetic analysis revealed the values of Km and Kcat as 0.6 μM and 2.5 min-1, respectively. Comparative protein modeling indicated an additional space in the substrate binding site of the parasitic enzyme compared with that of humans. Mutagenic analysis of the amino acid residue (Pro133) forming the additional space revealed a 5-fold increase in the wild-type Km value when replaced by a smaller (Ala) residue. Furthermore, catalytic activity was markedly affected by introducing a larger residue (Phe), thus creating the potential to develop a specific inhibitor of PfAp4A hydrolase.

Recognition and catalytic hydrolysis of adenosine 5′-triphosphate by cadmium(II) and L-glutamic acid

Interactions among Cd2+, glutamic acid (Glu), and adenosine 5′-triphosphate (ATP) have been studied by potentiometric pH titration, IR, Raman, fluorescence, and NMR methods. In the Cd2+-ATP binary system, the main interaction sites are the α-, β-, and γ-phosphate groups, N-1, and/or N-7. Cd2+ binds to the N-1 site at relatively low pH and binds to the N-7 site of adenosine ring of ATP with increasing pH. In the Cd2+-Glu-ATP ternary system, ATP mainly binds to Cd2+ by the triphosphate chain. Oxygens of Glu coordinate with Cd2+ to form a complex to catalyze ATP hydrolysis. Hydrolysis of ATP catalyzed by the CdGlu complex was studied at pH 7.0 and 80°C by 31P-NMR spectrometry. Kinetics studies showed that the rate constant of ATP hydrolysis was 0.0199 min-1 in the ternary system, which is 9.9-fold faster than that in the ATP solution (2.01 10-3 min-1). Hydrolysis occurs through an addition-elimination reaction mechanism with Cd2+ regulating the recognition and catalytic hydrolysis of ATP; water participates in the hydrolysis reaction of ATP at different steps with different functions in the ternary system.

REVERSIBLE TRANSPHOSPHORYLATION AMONG ATP, ADP, AND AMP IN THE PRESENCE OF METHYLATED CYCLODEXTRIN

A transphosphorylation among AMP, ADP, and ATP in a neutral aqueous solution with MgCl2 was examined in the presence of heptakis-(2,6-dimethyl)-β-cyclodextrin or heptakis-(2,3,6-trimethyl)-β-cyclodextrin.Adding methylated cyclodextrins was found to facilitate both forward and reversed reaction according to Eq.2 with an equilibrium constant of approximate unity.

Chloroplast adenylate kinase from tobacco. Purification and partial characterization

A soluble isoform of adenylate kinase (AK, EC 2.7.4,3) from tobacco leaves (Nicotiana tabacum L.) was purified about 60-fold by a protocol using ammonium sulphate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. The purified protein was homogeneous, as judged by SDS-PAGE, IEF-PAGE and Mono Q ion exchange chromatography, and had a specific activity of 500 nkat mg-1. Its M(r) was determined as 28 000 and 30 000 by SDS-PAGE and gel filtration, respectively. It is therefore monomeric and belongs to the long-variant-type adenylate kinases. The isoelectric point of ca 4.45, as measured by IEF-PAGE and the elution profile of the Mono Q column, is characteristic for a chloroplast AK isoform. Like the chloroplast AK of maize, the activity with ATP/AMP as substrates was about two times higher than with ADP and the apparent K(m) was about 10- times higher for ATP/AMP than for ADP. In contrast to the maize enzyme and many other eukaryotic AKs, both substrate binding sites showed an exceptionally high specificity for all three adenylate substrates, together with a rather low affinity, as judged by the apparent K(m)-values. These differences at the substrate-binding sites are confirmed by a low sensitivity of the enzyme to the competitive AK inhibitor diadenosine pentaphosphate, i.e. high K(i)-values.

Soluble expression in Escherichia coli of active human cyclic nucleotide phosphodiesterase isoform 4B2 in fusion with maltose-binding protein

Recombinant expression in Escherichia coli of human cyclic nucleotide phosphodiesterase 4B2 (hPDE4B2) fused to maltose-binding-protein (MBP-hPDE4B2) was investigated. hPDE4B2 DNA amplified via nested RT-PCR with total RNAs from U937 cells was ligated with pMAL-p2x. After induction at 18 °C for 16 h, soluble MBP-hPDE4B2 was produced in E. coli. MBP-hPDE4B2 after amylose-resin chromatography showed 35% homogeneity, and its Michaelis-Menten constant was 10 ± 2 μM (n = 3). Rolipram had a dissociation constant of 9 ± 2 nM (n = 2), and zinc ion was a potent inhibitor. Hence, MBP-hPDE4B2 was expressed in E. coli as a soluble active protein.

Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3

We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca 2+>Mg2+) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis-Menten kinetics with apparent Kms of 11, 10, 19 and 27 μM, respectively. In agreement with the Km values, the pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.

Enormous Acceleration by Cerium(IV) for the Hydrolysis of Nucleoside 3',5'-Cyclic Monophosphates at pH 7

At pH 7 and 30 deg C, 3',5'-cyclic monophosphates of adenosine and guanosine are promptly hydrolysed by Ce(NH4)2(NO3)6 (10-2 mol dm-3), with half-lives of 7 and 16 s, respectively.

Nucleoside conjugates V: Synthesis and biological activity of 9-(β-D-arabinofuranosyl)adenine conjugates of corticosteroids

Eight 5'-(steroid-21-phosphoryl)-9-(β-D-arabinofuranosyl)adenines (IV-XI) have been prepared and evaluated against L1210 lymphoid leukemia in culture. These include the 9-(β-D-arabinofuranosyl)adenine conjugates of hydrocortisone (IV), cortisone (V), corticosterone (VI), cortexolone (VII), 11-deoxycorticosterone (VIII), prednisolone (IX), prednisone (X), and dexamethasone (XI). Conjugates IV, IX, X, and XI inhibited the in vitro growth of L1210 lymphoid leukemia cells by 50% (ED50) at a concentration of 2.3-7.8 μM, while 9-(β-D-arabinofuranosyl)adenine (vidarabine, I) and its 5'-monophosphate (II) each showed ED50 value of 30 μM. All of the conjugates were enzymatically hydrolyzed to the corresponding steroid and II, the latter undergoing further hydrolysis to I, by phosphodiesterase I, 5'-nucleotidase, and acid phosphatase. However, these conjugates were resistant to hydrolysis by alkaline phosphatase and adenosine deaminase.

A combined experimental and theoretical study of the pH-dependent binding mode of NAD+ by water-soluble molecular clips

The highly selective recognition process of NAD+ and NADH (as important cofactors of many redox enzymes) by molecular clips in aqueous solution is studied systematically by a combined experimental and quantum-chemical approach. The strongly pH-

Synthesis and DNA cleavage activity of triazacrown-anthraquinone conjugates

1,4,7-Triazacyclononane (TACN), with DNA cleaving ability, was appended to anthraquinone via different spacers to construct the new compounds 1,8-[2,2′-(1,4,7-triazacyclonon)diethoxy] anthracene-9,10-dione hydrochloride (1) and 1,8-[2,2′-(1,4,7-triazacyclonon)dihexyloxy] anthracene-9,10-dione hydrochloride (2) as new agents for metal-free DNA cleavage. Fluorescence and CD spectroscopic studies suggest an intercalating DNA binding mode, and the apparent DNA binding constants of 1 and 2 are 3.93 × 107 and 6.07 × 107 M-1, respectively. Compound 2, bearing the longer spacer, exhibits the higher DNA binding ability. The apparent initial first-order rate constant (k obs) of DNA cleavage promoted by 1 and 2 (0.05 mM) in physiological media are 0.077 ± 0.0028 and 0.123 ± 0.0027 h-1, respectively. The 51-fold and 82-fold rate accelerations over parent TACN (the kobs is 0.0015 ± 0.00003 h-1 (0.05 mM) under the same conditions) are due to the anthraquinone moiety of compounds 1 and 2 intercalating into the DNA base pairs via stacking interactions. ESI-MS analysis of the dinucleotide cleavage promoted by 1 and 2, and radical scavenger inhibition studies suggest that the cleavage process is a hydrolytic mechanism.

Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and l-luciferin

The inhibition mechanisms of the firefly luciferase (Luc) by three of the most important inhibitors of the reactions catalysed by Luc, dehydroluciferyl-coenzyme A (L-CoA), dehydroluciferin (L) and l-luciferin (l-LH2) were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 μM) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and d-luciferin (d-LH2, from 3.75 up to 120 μM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that L-CoA is a non-competitive inhibitor of Luc (Ki = 0.88 ± 0.03 μM), L is a tight-binding uncompetitive inhibitor (Ki = 0.00490 ± 0.00009 μM) and l-LH2 acts as a mixed-type non-competitive-uncompetitive inhibitor (Ki = 0.68 ± 0.14 μM and αKi = 0.34 ± 0.16 μM). The Km values obtained for L-CoA, L and l-LH2 were 16.1 ± 1.0, 16.6 ± 2.3 and 14.4 ± 0.96 μM, respectively. L and l-LH2 are strong inhibitors of Luc, which may indicate an important role for these compounds in Luc characteristic flash profile. L-CoA Ki supports the conclusion that CoA can stimulate the light emission reaction by provoking the formation of a weaker inhibitor.

Reactivities and Site-Selectivities of Hydrolyses of ATP and UTP Promoted by Metal Complexes of Adenine-Linked Di-2-pyridylamine Derivatives

The reactivities and the site-selectivities of the hydrolyses of ATP and UTP by the catalysts of the metal complexes of adenine-linked di-2-pyridylamine ligands, (Py)2N-(CH2)n-Ade (L: n = 3, 4, 5, and 6), were examined. It was found that these adenine-dipyridylamine coordinated Cu2+ complexes (with 2:1 ratios of [CuII(L)]2+ : ATP or [CuII(L)]2+ : UTP) were more reactive for the hydrolyses of ATP and UTP than the complexes of ligands containing other metal ions (Mg2+, Ni2+, and Zn2+) at 40°C and pH 7.3 (HEPES buffer), as reflected in much higher product ratios of ADP/AMP and UDP/UMP than those of Cu2+ alone. The observed high reactivity and selectivity are interpreted in terms of the base-base stackings between an adenine moiety of ATP or an uracil moiety of UTP and an adenine of the ligands, and of the selective coordination of Cu2+ to oxide ions in phosphate residues in the ternary complexes of ligand-Cu2+-ATP. The Cu2+-complex of di-2-pyridylamine having no adenine moiety, which is the active center of [CuII(L)]2+, promoted the hydrolyses of ATP and UTP less efficiently than the aquacopper(II) ion. The number of spacer methylene groups of the ligands influenced the hydrolytic activity of the Cu2+-complexes of these adenine-dipyridylamine ligands. The complexes of [CuII(L-4)]2+ and [CuII(L-5)]2+ were the most reactive and site-selective for the hydrolyses of ATP and UTP, respectively.

Efficient Molecular Catalysis of ATP-Hydrolysis by Protonated Macrocyclic Polyamines

Molecular catalysis of ATP-hydrolysis by a number of protonated macrocyclic polyamines 1-9 has been investigated by 31P-NMR spectroscopy, and marked rate enhancements have been obtained.The largest acceleration is produced by the -N6O2 macrocycle 1, and the process displays the following properties: 1. protonated 1 forms very stable complexes with ATP, as well as with ADP and AMP; 2. it enhances the rate of ATP-hydrolysis by a factor of 103 at pH=8.5; the rate of hydrolysis is constant over a wide pH-range, from pH=2.5 to 8.5; 3. 1 is more efficient than acyclic analogues; 4. the products of the reaction are ortho phosphate (OP) and ADP, which is subsequently hydrolyzed to OP and AMP at a slower rate; 5. at pH>6.5, a transient species is detected, which is tentatively identified as a phosphoramidate intermediate, resulting from phosphorylation of the macrocycle 1; 6. the reaction presents first-order kinetics and is catalytic.The mechanism of the process is discussed in terms of initial formation of a complex between ATP and protonated 1, followed by an intracomplex reaction which may involve a combination of nucleophilic or acid catalysis with electrostatic catalysis.

Structure-reactivity relationship for the cobalt(III) complex-catalysed hydrolysis of adenosine 3′,5′-cyclic monophosphate

Hydrolysis of adenosine 3′,5′-cyclic monophosphate (cAMP) by cobalt(III) complexes [Co(N4XH2O)2]3+ (N4: two diamines or one tetraamine) has been systematically studied at pH 7 and 50°C. Both the catalytic activity and the product distribution are highly dependent on the nature of the amine ligand. The relative catalytic activities are cyclen (4000) > trien (500) > (tme)2 (57) > tren (37) > (tn)2 (22) > 2,3,2-tet (7) > (en)2 (1) ? cyclam, cth, dien. The pseudo-first-order rate constant for the cyclen complex (0.05 M) is 1.2 h-1 (half-life 35 min), corresponding to a 1010-fold acceleration with respect to the uncatalysed reaction. Of the two P-O linkages in cAMP, the cyclen, the trien and the 2,3,2-tet complexes preferentially cleave the P-O(5′) linkage, whereas the (tme)2 and the (tn)2 complexes promote P-O(3′) scission. Adenosine is the main product for hydrolysis by the (tme)2 complex, whereas adenosine monophosphates as the hydrolysis intermediates are accumulated in the catalysis by the trien complex.

Chemical synthesis and firefly luciferase produced dehydroluciferyl- coenzyme A

Dehydroluciferyl-coenzyme A (L-CoA) was chemically synthesized and characterized by MS, UV-vis spectrometry and RP-HPLC. The identity of the chemically synthesized compound with the one that was produced by firefly luciferase was confirmed. Moreover, the reversibility of the enzymatic conversion of dehydroluciferin?dehydroluciferyl-adenylate?L-CoA was also confirmed. The chemical synthesis of L-CoA, described here, may help the clarification of the activator effect of CoA on luciferase bioluminescent assays, in which the enzyme catalyzed formation of L-CoA and the consequent destruction of L-AMP is one of the possible explanations for that effect.

Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence

Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.

Pyrophosphate Formation via a Phosphoramidate Intermediate in Polyammonium Macrocycle/Metal Ion-Catalyzed Hydrolysis of ATP

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A STEREOCHEMICAL AND POSITIONAL ISOTOPE EXCHANGE STUDY OF THE MECHANISM OF ACTIVATION OF TYROSINE BY TYROSYL-tRNA SYNTHETASE FROM BACILLUS STEAROTHERMOPHILUS

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus catalyses the activation of (18)O2-tyrosine by adenosine 5'triphosphate with inversion of configuration at Pα.It also catalyses positional isotope exchange in adenosine 5'triphosphate in the presence of tyrosine, but not in its absence or in the presence of the competitive inhibitor tyrosinol.Together these results imply that the enzyme catalyses an associative "in line" displacement of pyrophosphate from Pα of ATP by tyrosine.

cUMP hydrolysis by PDE3A

As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a KM value of ~143?μM and a Vmax value of ~42?μmol/min/mg. PDE3A hydrolyzed cAMP with a KM value of ~0.7?μM and a Vmax of ~1.2?μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (Ki?=?57?nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.

Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5′-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP)

Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5′-phosphosulfate (APS or SO4-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed.

Dynamic Exchange of Substituents in a Prebiotic Organocatalyst: Initial Steps towards an Evolutionary System

All evolutionary biological processes lead to a change in heritable traits over successive generations. The responsible genetic information encoded in DNA is altered, selected, and inherited by mutation of the base sequence. While this is well known at the biological level, an evolutionary change at the molecular level of small organic molecules is unknown but represents an important prerequisite for the emergence of life. Here, we present a class of prebiotic imidazolidine-4-thione organocatalysts able to dynamically change their constitution and potentially capable to form an evolutionary system. These catalysts functionalize their building blocks and dynamically adapt to their (self-modified) environment by mutation of their own structure. Depending on the surrounding conditions, they show pronounced and opposing selectivity in their formation. Remarkably, the preferentially formed species can be associated with different catalytic properties, which enable multiple pathways for the transition from abiotic matter to functional biomolecules.

Synthesis method and application of vidarabine monophosphate

The invention belongs to the field of medicine synthesis, and discloses a synthesis method and application of vidarabine monophosphate. According to the synthesis method, 5-iodo-2-((phosphonooxy) methyl)-4-(tosyloxy)tetrahydrofuran-3-yl acetate and tert-butyl (8-hydroxy-9H-purin-6-yl)carbamate are subjected to condensation, epoxidation, ring opening and desulfurization reaction, and the vidarabine monophosphate is synthesized. According to the synthesis method of vidarabine monophosphate, provided by the invention, the industrial production steps are further simplified, the total reaction yield is improved, and the industrial production cost is reduced. The method is suitable for synthesizing vidarabine monophosphate, and the synthesized vidarabine monophosphate is used for preparing vidarabine monophosphate for injection.