24937-83-5Relevant academic research and scientific papers
Characterization of acetyl-CoA synthetase kinetics and ATP-binding
Gallego-Jara, Julia,Terol, Gema Lozano,écija Conesa, Ana,Zambelli, Barbara,Cánovas Díaz, Manuel,de Diego Puente, Teresa
, p. 1040 - 1049 (2019)
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
Wei, Li,Hu, Hong-Wen,Lu, Guo-Yuan,Shao, Ying,Zhou, Mi
, p. 8484 - 8492,9 (2012)
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
Chatterjee, Abhishek,Jurgenson, Christopher T.,Schroeder, Frank C.,Ealick, Steven E.,Begley, Tadhg P.
, p. 7158 - 7159 (2006)
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
Substrate Specificity and Chemical Mechanism for the Reaction Catalyzed by Glutamine Kinase
Taylor, Zane W.,Chamberlain, Alexandra R.,Raushel, Frank M.
, (2018)
Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (l-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of l-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of l-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated (Enz-X-Pβ-Pγ) and phosphorylated (Enz-X-Pβ) intermediates. In the absence of l-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within β-[18O4]-ATP in support of the formation of the Enz-X-Pβ-Pγintermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between l-glutamine phosphate and [15N-amide]-l-glutamine in direct support of the Enz-X-Pβintermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of d-glutamine, γ-l-glutamyl hydroxamate, γ-l-glutamyl hydrazide, and β-l-aspartyl hydroxamate, in addition to l-glutamine.
Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues
Chen, Jesse J.,Tsu, Christopher A.,Gavin, James M.,Milhollen, Michael A.,Bruzzese, Frank J.,Mallender, William D.,Sintchak, Michael D.,Bump, Nancy J.,Yang, Xiaofeng,Ma, Jingya,Loke, Huay-Keng,Xu, Qing,Li, Ping,Bence, Neil F.,Brownell, James E.,Dick, Lawrence R.
, p. 40867 - 40877 (2011)
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.
Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors
Florea, Mara,Nau, Werner M.
, p. 1033 - 1039 (2010)
A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-γ-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (μM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni2+, Mg2+, Mn2+, and Ca2+). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP). The Royal Society of Chemistry.
Rhodamine-based fluorescent probe for sequential detection of Al3+ ions and adenosine monophosphate in water
Kaur, Rajinder,Saini, Sanjeev,Kaur, Navneet,Singh, Narinder,Jang, Doo Ok
, (2020)
Organic nanoparticles (N1) were prepared by dispersing thiophene-conjugated rhodamine derivative 1 in a buffer solution (10 mM TRIS, pH 7.4, containing 1% DMSO, v/v). N1 selectively recognized Al3+ ions through the “OFF-ON” switching mechanism of the spirolactam ring in rhodamine. The resulting N1·Al3+ complex recognized the biologically important molecule adenosine monophosphate (AMP) through a cation displacement process with a detection limit of 2 nM. N1 was capable of determining the concentration of Al3+ ions in environmental and biological samples. Portable test strips of N1 were prepared for the recognition of Al3+ ions and AMP for practical uses. Furthermore, it was demonstrated that the N1·Al3+ complex facilitated real-time monitoring of AMP concentration in the hydrolysis of ATP and ADP.
Molecular characterization and mutational analysis of recombinant diadenosine 5′,5″-P1,P4-tetraphosphate hydrolase from Plasmodium falciparum
Osman, Waleed,Endo, Satoshi,Oh-hashi, Kentaro,Kitamura, Yoshiaki,Kitade, Yukio
, p. 1191 - 1196 (2012)
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.
pH-dependence in the hydrolytic action of the human fragile histidine triad
Huang, Kaisheng,Arabshahi, Abolfazl,Frey, Perry A.
, p. 5198 - 5206 (2005)
The human fragile histidine triad protein (Fhit) is a member of the HIT family of enzymes, which catalyze hydrolysis or nucleotidyltransfer reactions of dinucleoside polyphoshates. Fhit catalyzes the magnesium ion-dependent hydrolysis of P1-5′-O-adenosine-P3-5′-O- adenosine triphosphate (Ap3A) to adenosine-5′-O-phosphate (AMP) and the magnesium complex of adenosine-5′-O-diphosphate (ADP) by a double displacement mechanism, with the formation of an adenylyl enzyme as an intermediate. Fhit also catalyzes the hydrolysis of adenosine-5′- phosphoimidazolide (AMP-Im) and adenosine-5′-phospho-N-methylimidazolide (AMP-N-MeIm). The pH-dependence of these reactions is reported herein, in which the principal conclusions are as follows: The action of wild-type Fhit on MgAp3A is diffusion-limited and on AMP-Im and AMP-N-MeIm largely diffusion-limited and largely pH-independent. The actions of specifically mutated H96G-Fhit on AM-Im, and on AMP-N-MeIm, are not diffusion-limited and are pH-dependent. The actions of mutated forms of Fhit, H94G-Fhit, H98G-Fhit, and H94/98G-Fhit, are also not diffusion-limited and are pH-dependent. Log plots of kinetic parameters against pH show breaks that indicate a group on the enzyme must be protonated for maximal activity. Extensive analysis shows that the imidazole ring of His94 is not essential for the hydrolysis of MgAp3A or AMP-imidizolides, and the imidazole ring of His98 engages in binding the substrates. In the hydrolysis of AMP-Im, Fhit and its His94, His96, and His98 variants bind the monoanionic form of AMP-Im, and the proton required for formation of imidazole in the hydrolytic process originates with an acid/base group of the enzyme. Fhit and several variants also catalyze the hydrolysis of p-nitrophenyl-AMP. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.
Recognition and catalytic hydrolysis of adenosine 5′-triphosphate by cadmium(II) and L-glutamic acid
Zhou, Jin-Ying,Lu, Gong-Xuan
, p. 3441 - 3453 (2011)
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.
