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1
Willadsen, P., J. M. Nielsen, and G. A. Riding. "Purification and properties of a novel nucleotide-hydrolysing enzyme (5′-nucleotidase) from Boophilus microplus." Biochemical Journal 258, no. 1 (February 15, 1989): 79–85. http://dx.doi.org/10.1042/bj2580079.
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The tick Boophilus microplus contains a nucleoside phosphate-hydrolysing enzyme which, in many respects, resembles the well characterized 5'-nucleotidase from mammalian tissue. The tick enzyme has been purified to homogeneity. It is a membrane-bound glycoprotein with an apparent Mr of 67,000 and, although it fails to hydrolyse a range of nucleoside 2'- or 3'-monophosphates, it has broad specificity for the 5' derivatives. Further investigation of the enzyme's substrate specificity, however, shows some important differences from the mammalian nucleotidases. It hydrolyses both bis-p-nitrophenyl phosphate and p-nitrophenyl phenylphosphonate, typical substrates for phosphodiesterases. However, the tick enzyme is most strikingly different from the mammalian enzymes in that it hydrolyses not only AMP but ADP and ATP as well. Further, the products of the hydrolysis of ATP are adenosine and tripolyphosphate, a reaction which has not been reported previously. The products of ADP hydrolysis are adenosine and pyrophosphate.
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Dahlmann, B., L. Kuehn, and H. Reinauer. "Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle." Biochemical Journal 309, no. 1 (July 1, 1995): 195–202. http://dx.doi.org/10.1042/bj3090195.
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The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5′-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5′-[beta,gamma-imido]triphosphate, adenosine 5′-O-[gamma-thio]triphosphate, adenosine 5′-O-[beta-thio]-diphosphate and adenosine 5′-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli.
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ROCHA, J. B. T., C. F. Mello, J. J. F. Sarkis, and R. D. Dias. "Undernutrition during the preweaning period changes calcium ATPase and ADPase activities of synaptosomal fractions of weanling rats." British Journal of Nutrition 63, no. 2 (March 1990): 273–83. http://dx.doi.org/10.1079/bjn19900114.
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The presence of activities that hydrolyse externally added ATP to adenosine in synaptosomal preparations from various sources is well demonstrated. The hydrolysis of ATP to AMP can be mediated either by the concerted action of enzymes or by an ATP-diphosphohydrolase (EC 3.6.1.5; apyrase). Undernutrition during the preweaning period can delay the development of several enzymes involved in the metabolism of neurotransmitters or neuronal function. In young rats, the presence of an apyrase in synaptosomal preparations from cerebral cortex was investigated. The results suggested that the hydrolysis of externally added ATP and ADP can be mediated by a single enzyme. The effects of preweaning undernutrition on the hydrolysis of ATP and ADP were also investigated. In weanling rats, previous undernutrition caused a decrease of about 20% in the hydrolysis of both substrates in synaptosomal fractions.
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Petri, Jessica, Yoshio Nakatani, Martin G. Montgomery, Scott A. Ferguson, David Aragão, Andrew G. W. Leslie, Adam Heikal, John E. Walker, and Gregory M. Cook. "Structure of F1-ATPase from the obligate anaerobeFusobacterium nucleatum." Open Biology 9, no. 6 (June 2019): 190066. http://dx.doi.org/10.1098/rsob.190066.
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The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from the pathogenic anaerobic bacteriumFusobacterium nucleatum. The enzyme can hydrolyse ATP but is partially inhibited. The structure is similar to those of the F1-ATPases fromCaldalkalibacillus thermarum, which is more strongly inhibited in ATP hydrolysis, and inMycobacterium smegmatis, which has a very low ATP hydrolytic activity. The βE-subunits in all three enzymes are in the conventional ‘open’ state, and in the case ofC. thermarumandM. smegmatis, they are occupied by an ADP and phosphate (or sulfate), but inF. nucleatum, the occupancy by ADP appears to be partial. It is likely that the hydrolytic activity of theF. nucleatumenzyme is regulated by the concentration of ADP, as in mitochondria.
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Buchet, Rene, Camille Tribes, Valentine Rouaix, Bastien Doumèche, Michele Fiore, Yuqing Wu, David Magne, and Saida Mebarek. "Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi." International Journal of Molecular Sciences 22, no. 6 (March 14, 2021): 2948. http://dx.doi.org/10.3390/ijms22062948.
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(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.
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Marquenet, Emélie, and Evelyne Richet. "Conserved Motifs Involved in ATP Hydrolysis by MalT, a Signal Transduction ATPase with Numerous Domains from Escherichia coli." Journal of Bacteriology 192, no. 19 (August 6, 2010): 5181–91. http://dx.doi.org/10.1128/jb.00522-10.
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ABSTRACT The signal transduction ATPases with numerous domains (STAND) are sophisticated signaling proteins that are related to AAA+ proteins and control various biological processes, including apoptosis, gene expression, and innate immunity. They function as tightly regulated switches, with the off and on positions corresponding to an ADP-bound, monomeric form and an ATP-bound, multimeric form, respectively. Protein activation is triggered by inducer binding to the sensor domain. ATP hydrolysis by the nucleotide-binding oligomerization domain (NOD) ensures the generation of the ADP-bound form. Here, we use MalT, an Escherichia coli transcription activator, as a model system to identify STAND conserved motifs involved in ATP hydrolysis besides the catalytic acidic residue. Alanine substitution of the conserved polar residue (H131) that is located two residues downstream from the catalytic residue (D129) blocks ATP hydrolysis and traps MalT in an active, ATP-bound, multimeric form. This polar residue is also conserved in AAA+. Based on AAA+ X-ray structures, we proposed that it is responsible for the proper positioning of the catalytic and the sensor I residues for the hydrolytic attack. Alanine substitution of the putative STAND sensor I (R160) abolished MalT activity. Substitutions of R171 impaired both ATP hydrolysis and multimerization, which is consistent with an arginine finger function and provides further evidence that ATP hydrolysis is primarily catalyzed by MalT multimers.
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Sarkis, J. J. F., J. A. Guimarães, and J. M. C. Ribeiro. "Salivary apyrase of Rhodnius prolixus. Kinetics and purification." Biochemical Journal 233, no. 3 (February 1, 1986): 885–91. http://dx.doi.org/10.1042/bj2330885.
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The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5′-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.
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Ferguson, Scott A., Gregory M. Cook, Martin G. Montgomery, Andrew G. W. Leslie, and John E. Walker. "Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum." Proceedings of the National Academy of Sciences 113, no. 39 (September 12, 2016): 10860–65. http://dx.doi.org/10.1073/pnas.1612035113.
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The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.
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Krah, Alexander, Mariel Zarco-Zavala, and Duncan G. G. McMillan. "Insights into the regulatory function of the ɛ subunit from bacterial F-type ATP synthases: a comparison of structural, biochemical and biophysical data." Open Biology 8, no. 5 (May 2018): 170275. http://dx.doi.org/10.1098/rsob.170275.
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ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found in living cells. These enzymes are driven by an electrochemical ion gradient, which allows the catalytic evolution of ATP by a binding change mechanism. Most ATP synthases are capable of catalysing ATP hydrolysis to varying degrees, and to prevent wasteful ATP hydrolysis, bacteria and mitochondria have regulatory mechanisms such as ADP inhibition. Additionally, ɛ subunit inhibition has also been described in three bacterial systems, Escherichia coli , Bacillus PS3 and Caldalkalibacillus thermarum TA2.A1. Previous studies suggest that the ɛ subunit is capable of undergoing an ATP-dependent conformational change from the ATP hydrolytic inhibitory ‘extended’ conformation to the ATP-induced non-inhibitory ‘hairpin’ conformation. A recently published crystal structure of the F 1 domain of the C. thermarum TA2.A1 F 1 F o ATP synthase revealed a mutant ɛ subunit lacking the ability to bind ATP in a hairpin conformation. This is a surprising observation considering it is an organism that performs no ATP hydrolysis in vivo , and appears to challenge the current dogma on the regulatory role of the ɛ subunit. This has prompted a re-examination of present knowledge of the ɛ subunits role in different organisms. Here, we compare published biochemical, biophysical and structural data involving ɛ subunit-mediated ATP hydrolysis regulation in a variety of organisms, concluding that the ɛ subunit from the bacterial F-type ATP synthases is indeed capable of regulating ATP hydrolysis activity in a wide variety of bacteria, making it a potentially valuable drug target, but its exact role is still under debate.
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COIMBRA, E. S., S. C. GONÇALVES-DA-COSTA, S. CORTE-REAL, F. G. R. DE FREITAS, A. C. DURÃO, C. S. F. SOUZA, M. I. SILVA-SANTOS, and E. G. VASCONCELOS. "Characterization and cytochemical localization of an ATP diphosphohydrolase from Leishmania amazonensis promastigotes." Parasitology 124, no. 2 (February 2002): 137–43. http://dx.doi.org/10.1017/s0031182001001056.
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An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6·5–8·5 were examined using ATP or p-NPP as substrate. At pH 7·4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5–10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.
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WU, Xueji, Mihiro YANO, Hiroyo WASHIDA, and Hiroshi KIDO. "The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis." Biochemical Journal 378, no. 3 (March 15, 2004): 793–99. http://dx.doi.org/10.1042/bj20031680.
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The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP–ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403–414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis.
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Jackson, A. P., and A. Maxwell. "Identifying the catalytic residue of the ATPase reaction of DNA gyrase." Proceedings of the National Academy of Sciences 90, no. 23 (December 1, 1993): 11232–36. http://dx.doi.org/10.1073/pnas.90.23.11232.
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We propose a mechanism for the hydrolysis of ATP by the DNA gyrase B protein in which Glu42 acts as a general base and His38 has a role in aligning and polarizing the glutamate residue. We have tested this mechanism by site-directed mutagenesis, converting Glu42 to Ala, Asp, and Gln, and His38 to Ala. In the presence of wild-type A protein, B proteins bearing the mutations Ala42 and Gln42 show no detectable supercoiling or ATPase activities, while Asp42 and Ala38 proteins have reduced activities. In the DNA cleavage and relaxation reactions of gyrase, which do not require ATP hydrolysis, wild-type and mutant proteins have similar activities. When the 43-kDa N-terminal fragment of the gyrase B protein (which hydrolyzes ATP) contained the mutations Ala42 or Gln42, ATP was bound but not hydrolyzed, supporting the idea that Glu42 is involved in hydrolysis but not nucleotide binding.
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Chen, Wei, and Guido Guidotti. "Soluble Apyrases Release ADP during ATP Hydrolysis." Biochemical and Biophysical Research Communications 282, no. 1 (March 2001): 90–95. http://dx.doi.org/10.1006/bbrc.2001.4555.
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DELGADO, Jerónimo, Gloria MORO, Ana SABORIDO, and Alicia MEGÍAS. "T-tubule membranes from chicken skeletal muscle possess an enzymic cascade for degradation of extracellular ATP." Biochemical Journal 327, no. 3 (November 1, 1997): 899–907. http://dx.doi.org/10.1042/bj3270899.
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The chicken T-tubule Mg2+-ATPase is an integral membrane glycoprotein that presents properties different from those of other ATPases located in skeletal muscle cells and exhibits ATP-hydrolysing activity on the extracellular side of the transverse tubule (TT) membranes. In this study we demonstrate that TT vesicles purified from chicken skeletal muscle possess ecto-ADPase and ecto-5ʹ-nucleotidase activities that, along with ecto-ATPase, are able to sequentially degrade extracellular ATP to ADP, AMP and adenosine. Characterization studies of these TT ectonucleotidases revealed remarkable differences between ecto-ATPase and ecto-ADPase activities with respect to thermal stability, temperature dependence of the hydrolytic activity, effect of ionic strength, kinetic behaviour, divalent cation preference and responses to azide, N-ethylmaleimide, NaSCN, Triton X-100 and concanavalin A. Ecto-ATPase, but not ecto-ADPase, was inhibited by a polyclonal antibody against the chicken TT ecto-ATPase. On the basis of these results we propose that ATP and ADP hydrolysis are accomplished by two distinct enzymes and therefore the TT ecto-ATPase is not an apyrase. 5ʹ-Nucleotidase activity was inhibited by adenosine 5ʹ-[α,β-methylene]diphosphate and concanavalin A, followed simple Michaelis-Menten kinetics and was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that AMP hydrolysis in T-tubules is catalysed by a typical ecto-5ʹ-nucleotidase. Results obtained from electrophoresis experiments under native conditions suggest that ecto-ATPase, ecto-ADPase and 5ʹ-nucleotidase might be associated, forming functional complexes in the T-tubule membranes. The TT ectonucleotidases constitute an enzymic cascade for the degradation of extracellular ATP that might be involved in the regulation of purinergic signalling in the muscle fibre.
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TASCA, T., C. D. BONAN, G. A. DE CARLI, J. J. F. SARKIS, and J. F. ALDERETE. "Heterogeneity in extracellular nucleotide hydrolysis among clinical isolates ofTrichomonas vaginalis." Parasitology 131, no. 1 (March 7, 2005): 71–78. http://dx.doi.org/10.1017/s0031182005007377.
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Trichomonas vaginalisis a parasitic protozoan that causes trichomonosis, a sexually-transmitted disease, with serious sequelae to women and men. As the host–parasite relationship is complex, it is important to investigate biochemical aspects of the parasite that contribute to our understanding of trichomonal biology and pathogenesis. Nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1), which hydrolyses extracellular ATP and ADP, and ecto-5′-nucleotidase, which hyrolyses AMP, have been characterized in laboratory isolates ofT. vaginalis. Here we show that the extracellular ATP[ratio ]ADP hydrolysis ratio varies among fresh clinical isolates, which presented higher ATPase and ADPase activities than long-term-grown isolates. Growth of parasites in iron-replete and iron-depleted medium resulted in different, albeit minor, patterns in extracellular ATP and ADP hydrolysis among isolates. Importantly, some isolates had low or absent ecto-5′-nucleotidase activity, regardless of environmental conditions tested. For isolates with ecto-5′-nucleotidase activity, high- and low-iron trichomonads had increased and decreased levels of activity, respectively, compared to organisms grown in normal TYM-serum medium. This suggests a regulation in expression of either the enzyme amounts and/or activity under the control of iron. Finally, we found no correlation between the presence or absence of dsRNA virus infection among trichomonad isolates and NTPDase and ecto-5′-nucleotidase activities.
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Sharma, Susan, and Amy L. Davidson. "Vanadate-Induced Trapping of Nucleotides by Purified Maltose Transport Complex Requires ATP Hydrolysis." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6570–76. http://dx.doi.org/10.1128/jb.182.23.6570-6576.2000.
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ABSTRACT The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK2 transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl2 or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.
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Warburton, D., S. Buckley, and L. Cosico. "P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes." Journal of Applied Physiology 66, no. 2 (February 1, 1989): 901–5. http://dx.doi.org/10.1152/jappl.1989.66.2.901.
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Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3′,5′-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5′-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.
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Meghji, P., J. D. Pearson, and L. L. Slakey. "Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart." Biochemical Journal 308, no. 3 (June 15, 1995): 725–31. http://dx.doi.org/10.1042/bj3080725.
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We have characterized the ectonucleotidases that catalyse the reaction sequence ATP-->ADP-->AMP-->adenosine on microvascular endothelial cells cultured from the rat heart. Computer simulation and data fitting of progress of reaction curves showed that depletion of substrate at the cell surface dominates the regulation of the rate of hydrolysis of ATP when it is presented to the cells. Preferential delivery of AMP by ADPase to 5′-nucleotidase makes a significant contribution to the regulation of adenosine production from ATP or ADP. By contrast, we found no evidence for the preferential delivery of ADP from ATPase to ADPase. Feed-forward inhibition of AMP hydrolysis by ADP and/or ATP also modulated the rate of adenosine production. The properties of the ectonucleotidases on rat heart microvascular cells are such that adenosine is produced at a steady rate over a wide range of ATP concentrations.
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Ogilvie, A., J. Lüthje, U. Pohl, and R. Busse. "Identification and partial characterization of an adenosine(5′)tetraphospho(5′)adenosine hydrolase on intact bovine aortic endothelial cells." Biochemical Journal 259, no. 1 (April 1, 1989): 97–103. http://dx.doi.org/10.1042/bj2590097.
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The biologically active dinucleotides adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and adenosine(5′)-triphospho(5′)adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.
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Vetri, Francesco, Haoliang Xu, Lizhen Mao, Chanannait Paisansathan, and Dale A. Pelligrino. "ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 4 (October 2011): H1369—H1377. http://dx.doi.org/10.1152/ajpheart.00556.2011.
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ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5′-nucleotidase inhibitor α,β-methylene adenosine 5′-diphosphate (AOPCP), 2) the A2 receptor blocker ZM 241385, 3) the ADP P2Y1 receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 μM, but not 100 μM, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 μM ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A2 receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y1 activation.
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Williams, Nicholas H. "Magnesium Ion Catalyzed ATP Hydrolysis." Journal of the American Chemical Society 122, no. 48 (December 2000): 12023–24. http://dx.doi.org/10.1021/ja0013374.
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Korn, E., M. Carlier, and D. Pantaloni. "Actin polymerization and ATP hydrolysis." Science 238, no. 4827 (October 30, 1987): 638–44. http://dx.doi.org/10.1126/science.3672117.
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Classen, J. Barthelow, Wolfgang J. Mergner, and M. Costa. "ATP hydrolysis by ischemic Mitochondria." Journal of Cellular Physiology 141, no. 1 (October 1989): 53–59. http://dx.doi.org/10.1002/jcp.1041410109.
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Carlier, M. "Actin polymerization and ATP hydrolysis." Advances in Biophysics 26 (1990): 51–73. http://dx.doi.org/10.1016/0065-227x(90)90007-g.
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Martins, Samantha M., Christiane R. Torres, and Sérgio T. Ferreira. "Inhibition of the Ecto-ATPdiphosphohydrolase of Schistosoma mansoni by Thapsigargin." Bioscience Reports 20, no. 5 (October 1, 2000): 369–81. http://dx.doi.org/10.1023/a:1010330017583.
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ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable ofhydrolyzing nucleoside di- and triphosphates. Although a number ofpossible physiological roles have been proposed for ATPDases, detailedstudies on structure-function relationships have generally been hamperedby the lack of specific inhibitors of these enzymes. We have previouslycharacterized a Ca2+-activated ATPDase on the external surface ofthe tegument of Schistosoma mansoni, the etiologic agent of humanschistosomiasis. In the present work, we have examined the effectsof thapsigargin, a sesquiterpene lactone known as a high affinityinhibitor of sarco-endoplasmic reticulum calcium transport (SERCA)ATPase, on ATPDase activity. Whereas other lactones tested had littleor no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (Ki∼20 μM). Interestingly, hydrolysis of ADP was notinhibited by thapsigargin. The lack of inhibition of ATPase activityby orthovanadate, a specific inhibitor of P-type ATPases, and theinhibition of the Mg2+-stimulated ATP hydrolysis by thapsigarginruled out the possibility that the observed inhibition of the ATPDaseby thapsigargin could be due to the presence of contaminating SERCAATPases in our preparation. Kinetic analysis indicated that a singleactive site in the ATPDase is responsible for hydrolysis of both ATPand ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigarginwas little or not affected by changes in free Ca2+ or Mg2+concentrations. These results suggest that interaction of thapsigarginwith the S. mansoni ATPDase prevents binding of ATP or its hydrolysisat the active site, while ADP can still undergo catalysis.
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Kroll, K., D. J. Kinzie, and L. A. Gustafson. "Open-system kinetics of myocardial phosphoenergetics during coronary underperfusion." American Journal of Physiology-Heart and Circulatory Physiology 272, no. 6 (June 1, 1997): H2563—H2576. http://dx.doi.org/10.1152/ajpheart.1997.272.6.h2563.
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A novel hypothesis is proposed and tested describing open-system kinetics for myocardial phosphoenergetics. The hypothesis is that during severe coronary underperfusion there is precise matching of the rates of ATP synthesis and hydrolysis, but despite the precise balance of ATP rates, there is a decrease in the concentration of ATP and an increase in the concentration of phosphocreatine (PCr) caused by the hydrolysis of AMP to adenosine. Isolated rabbit hearts were perfused using a crystalloid medium, and coronary flow was reduced by 95% from baseline for 45 min followed by reperfusion. Phosphorus nuclear magnetic resonance spectroscopy showed a rapid decrease in PCr concentration to 25% of baseline at the onset of underperfusion followed by a gradual increase in PCr to 42% of baseline, while ATP decreased continuously to 65% of baseline. The kinetics of PCr and ATP could only be described by the precise matching of the rates of ATP synthesis and ATP hydrolysis and an open adenylate system that included the decrease in cytosolic AMP concentration via the production and efflux of adenosine. To confirm the hypothesis of open-system kinetics, two independent predictions were tested in separate experiments: 1) total coronary venous purine efflux (adenosine+inosine+hypoxanthine) during underperfusion was equal to the decrease in ATP concentration, and 2) there was no increase in PCr during moderate coronary underperfusion (80% flow reduction). In conclusion, the open nature of the myocardial adenylate system causes mass action effects that exert novel control over PCr and ATP concentrations during coronary underperfusion. The open-system kinetics cause ATP to decrease and PCr to increase, even though there is precise matching of the rates of ATP synthesis and hydrolysis. Finally, the hydrolysis of AMP to adenosine may benefit tissue survival during ischemia by improving the free energy of ATP hydrolysis, thereby delaying or preventing calcium overload.
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Greenleaf, William B., Jingping Shen, Dahai Gai, and Xiaojiang S. Chen. "Systematic Study of the Functions for the Residues around the Nucleotide Pocket in Simian Virus 40 AAA+ Hexameric Helicase." Journal of Virology 82, no. 12 (April 9, 2008): 6017–23. http://dx.doi.org/10.1128/jvi.00387-08.
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ABSTRACT The high-resolution structural data for simian virus 40 large-T-antigen helicase revealed a set of nine residues bound to ATP/ADP directly or indirectly. The functional role of each of these residues in ATP hydrolysis and also the helicase function of this AAA+ (ATPases associated with various cellular activities) molecular motor are unclear. Here, we report our mutational analysis of each of these residues to examine their functionality in oligomerization, DNA binding, ATP hydrolysis, and double-stranded DNA (dsDNA) unwinding. All mutants were capable of oligomerization in the presence of ATP and could bind single-stranded DNA and dsDNA. ATP hydrolysis was substantially reduced for proteins with mutations of residues making direct contact with the γ-phosphate of ATP or the apical water molecule. A potentially noncanonical “arginine finger” residue, K418, is critical for ATP hydrolysis and helicase function, suggesting a new type of arginine finger role by a lysine in the stabilization of the transition state during ATP hydrolysis. Interestingly, our mutational data suggest that the positive- and negative-charge interactions in the uniquely observed residue pairs, R498/D499 and R540/D502, in large-T-antigen helicase are critically involved in the transfer of energy of ATP binding/hydrolysis to DNA unwinding.
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Meghji, P., J. D. Pearson, and L. L. Slakey. "Regulation of extracellular adenosine production by ectonucleotidases of adult rat ventricular myocytes." American Journal of Physiology-Heart and Circulatory Physiology 263, no. 1 (July 1, 1992): H40—H47. http://dx.doi.org/10.1152/ajpheart.1992.263.1.h40.
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We have investigated the kinetic properties of the extracellular reaction sequence ATP----ADP----AMP----adenosine catalyzed by ectonucleotidases at the surface of adult rat cardiac myocytes. Analysis of progress of reaction curves indicates that depletion of substrate at cell surfaces dominates the regulation of the rate of hydrolysis of ATP or of ADP when it is the initial substrate. Preferential delivery of intermediate products to be substrates at cell surfaces makes a significant contribution to the regulation of adenosine production from ATP or ADP. Preferential delivery has more impact on the delivery of ADP from adenosinetriphosphatase (ATPase) to adenosinediphosphatase (ADPase) than on delivery of AMP from ADPase to 5'-nucleotidase. At high initial ATP concentrations, feed-forward inhibition of AMP hydrolysis also modulates the rate of adenosine production. Taken together, the properties of the ectonucleotidases on the myocyte provide a milieu at the cell surface that tends to be poor in nucleotides, especially ATP and ADP (P2 purinoceptor agonists), and rich in adenosine (a P1 purinoceptor agonist) during periods of supply of extracellular nucleotides.
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Klostermeier, Dagmar. "Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 611–16. http://dx.doi.org/10.1042/bst0390611.
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Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.
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Varghese, Febin, James N. Blaza, Andrew J. Y. Jones, Owen D. Jarman, and Judy Hirst. "Deleting the IF 1 -like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis." Open Biology 8, no. 1 (January 2018): 170206. http://dx.doi.org/10.1098/rsob.170206.
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In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF 1 . Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F 1 -ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.
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Tang, Liang, Haiyan Zhao, Theodore Christensen, Zihan Lin, and Annie Lynn. "Visualizing ATP hydrolysis in a viral DNA-packaging molecular motor." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1604. http://dx.doi.org/10.1107/s2053273314083958.
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Many DNA viruses encode powerful molecular machines to package viral genome into preformed protein shells. These DNA-packaging motors contain an ATPase module that converts the chemical reaction of ATP hydrolysis to physical motion of DNA. We previously determined the structures of the DNA-packaging motor gp2 of Shigella phage Sf6 in the apo form and in complex with ADP and ATP-gamma-S (Zhao et al, 2013, PNAS, 110, 8075). Here we report the structure of gp2 in complex with its substrate ATP at 2.0 Angstrom resolution. To our knowledge, this is the first time to capture, at high resolution, a precatalytic state for ASCE-superfamily ATPases, which include a large group of nucleic acid helicases and translocases involved in a broad range of cellular and viral processes. The structure reveals the precise architecture of the ATP-bound state of the motor immediately prior to catalysis. Comparison with structures of the apo and ADP-complexed forms unveils motions of the Walker A motif coupled with ATP and Mg2+ binding and ATP hydrolysis. In the Walker B motif, residue E118 undergoes a side chain conformational switching coupled with the ATP hydrolysis, whereas residue E119 locks residue R51 side chain to a conformation that is readily reachable to residue E118 side chain. Residue E121 in the Walker B motif deprotonates a water molecule, which acts as a nucleophile to attack the gamma-phosphorous, leading to ATP hydrolysis. The alpha-helix (residue G182-R194) in the linker domain undergoes a translational motion against the ATPase domain triggered by ATP hydrolysis, serving as a mechanism for translating the energy from the chemical reaction into physical movement of DNA. We further observed the time course of ATP hydrolysis by gp2 by determining structures of gp2:ATP complexes captured at various incubation time. These structures have made it possible to delineate, at atomic detail, the complete cycle of ATP hydrolysis of this viral DNA-packaging molecular motor.
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Laketa, Danijela, Ivana Bjelobaba, Danijela Stojkov, Irena Lavrnja, Ana Parabucki, Mirjana Stojiljkovic, and Nadezda Nedeljkovic. "Brain cortical injury induces changes in peripheral lymphocyte ectonucleotidase activities." Archives of Biological Sciences 65, no. 1 (2013): 33–42. http://dx.doi.org/10.2298/abs1301033l.
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Injury and other pathological conditions induce a massive release of ATP and ADP that initiate an immune response. Extracellular nucleotides are degraded by ectonucleotidases: enzymes from E-NTPDase and E-NPP families sequentially hydrolyze ATP and ADP to AMP, which is further hydrolyzed by ecto-5?-nucleotidase to adenosine that exerts suppressive effects on immune cells. We investigated the ectonucleotidase activities of peripheral lymphocytes at different post-injury times after an unilateral brain injury in the rat. Significant and dynamic changes in the lymphocytic ectonucleotidase activities were obtained. ATP- and ADP-hydrolysis changes, together with their calculated ratios, indicate the major contribution of E-NTPDase 1 and its comparable upregulation between sham operation and injury. AMP hydrolysis changes were more brain-injury specific, with a longer-lasting lymphocytic response induced by cortical stab injury (CSI). In summary, CSI and sham operation induce the upregulation of the whole enzyme chain for adenine nucleotide hydrolysis in lymphocytes, suggesting an important roles of ectonucleotidases in the course of recovery after brain injury.
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Soga, Naoki, Kazuya Kimura, Kazuhiko Kinosita, Masasuke Yoshida, and Toshiharu Suzuki. "Perfect chemomechanical coupling of FoF1-ATP synthase." Proceedings of the National Academy of Sciences 114, no. 19 (April 25, 2017): 4960–65. http://dx.doi.org/10.1073/pnas.1700801114.
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FoF1-ATP synthase (FoF1) couples H+ flow in Fo domain and ATP synthesis/hydrolysis in F1 domain through rotation of the central rotor shaft, and the H+/ATP ratio is crucial to understand the coupling mechanism and energy yield in cells. Although H+/ATP ratio of the perfectly coupling enzyme can be predicted from the copy number of catalytic β subunits and that of H+ binding c subunits as c/β, the actual H+/ATP ratio can vary depending on coupling efficiency. Here, we report actual H+/ATP ratio of thermophilic Bacillus FoF1, whose c/β is 10/3. Proteoliposomes reconstituted with the FoF1 were energized with ΔpH and Δψ by the acid−base transition and by valinomycin-mediated diffusion potential of K+ under various [ATP]/([ADP]⋅[Pi]) conditions, and the initial rate of ATP synthesis/hydrolysis was measured. Analyses of thermodynamically equilibrated states, where net ATP synthesis/hydrolysis is zero, show linear correlation between the chemical potential of ATP synthesis/hydrolysis and the proton motive force, giving the slope of the linear function, that is, H+/ATP ratio, 3.3 ± 0.1. This value agrees well with the c/β ratio. Thus, chemomechanical coupling between Fo and F1 is perfect.
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Unciuleac, Mihaela-Carmen, Aviv Meir, Chaoyou Xue, Garrett M. Warren, Eric C. Greene, and Stewart Shuman. "Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease." Proceedings of the National Academy of Sciences 118, no. 11 (March 8, 2021): e2023955118. http://dx.doi.org/10.1073/pnas.2023955118.
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Mycobacterial AdnAB is a heterodimeric helicase–nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3′ to 5′ translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet–like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB–DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.
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Sandall, Christina F., Bjoern K. Ziehr, and Justin A. MacDonald. "ATP-Binding and Hydrolysis in Inflammasome Activation." Molecules 25, no. 19 (October 7, 2020): 4572. http://dx.doi.org/10.3390/molecules25194572.
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The prototypical model for NOD-like receptor (NLR) inflammasome assembly includes nucleotide-dependent activation of the NLR downstream of pathogen- or danger-associated molecular pattern (PAMP or DAMP) recognition, followed by nucleation of hetero-oligomeric platforms that lie upstream of inflammatory responses associated with innate immunity. As members of the STAND ATPases, the NLRs are generally thought to share a similar model of ATP-dependent activation and effect. However, recent observations have challenged this paradigm to reveal novel and complex biochemical processes to discern NLRs from other STAND proteins. In this review, we highlight past findings that identify the regulatory importance of conserved ATP-binding and hydrolysis motifs within the nucleotide-binding NACHT domain of NLRs and explore recent breakthroughs that generate connections between NLR protein structure and function. Indeed, newly deposited NLR structures for NLRC4 and NLRP3 have provided unique perspectives on the ATP-dependency of inflammasome activation. Novel molecular dynamic simulations of NLRP3 examined the active site of ADP- and ATP-bound models. The findings support distinctions in nucleotide-binding domain topology with occupancy of ATP or ADP that are in turn disseminated on to the global protein structure. Ultimately, studies continue to reveal how the ATP-binding and hydrolysis properties of NACHT domains in different NLRs integrate with signaling modules and binding partners to control innate immune responses at the molecular level.
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Jih, Kang-Yang, Yoshiro Sohma, and Tzyh-Chang Hwang. "Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation." Journal of General Physiology 140, no. 4 (September 10, 2012): 347–59. http://dx.doi.org/10.1085/jgp.201210834.
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Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR’s gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating—a strict coupling between the ATP hydrolysis cycle and the gating cycle—is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1→O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1→O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.
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OLIVEIRA, CAMILA B., ALEKSANDRO S. DA SILVA, VIVIANE C. G. SOUZA, MARCIO M. COSTA, JEANDRE A. S. JAQUES, DANIELA B. R. LEAL, SONIA T. A. LOPES, and SILVIA G. MONTEIRO. "NTPDase activity in lymphocytes of rats infected by Trypanosoma evansi." Parasitology 139, no. 2 (January 5, 2012): 232–36. http://dx.doi.org/10.1017/s0031182011001879.
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SUMMARYTrypanosoma evansi is the aetiological agent of trypanosomosis in domestic animals. In this pathology, an inflammatory response can be observed and, as a consequence, the increase of extracellular adenine nucleotides such as ATP. These nucleotide concentrations are regulated by ectoenzymes such as NTPDase (EC 3.6.1.5, CD39), which catalyses the hydrolysis of ATP and ADP into AMP. In this study, the activity of NTPDase in lymphocytes of rats experimentally infected with T. evansi was evaluated. The animals were inoculated with the parasite and monitored by blood smear on a daily basis. The animals were then were divided into 4 groups according to the degree of parasitaemia and period of infection. The blood collections for enzyme analysis and lymphocyte count were performed on the 3rd (beginning of infection), 5th (acute infection) and 15th (chronic infection) days post-infection (p.i.). The control group was composed of non-infected animals. In the infected group a decrease in ATP hydrolysis (36%) was observed on the 3rd day p.i. and a decrease in ADP hydrolysis (62%) was observed on the 5th day p.i. when compared to the control. On the 15th day p.i., an increase in ATP (94%) and ADP (50%) hydrolysis was observed in the infected group. Considering these data it is suggested that NTPDase activity is altered on the surface of lymphocytes of rats infected with T. evansi at different time-points of infection.
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Jih, Kang-Yang, Yoshiro Sohma, Min Li, and Tzyh-Chang Hwang. "Identification of a novel post-hydrolytic state in CFTR gating." Journal of General Physiology 139, no. 5 (April 16, 2012): 359–70. http://dx.doi.org/10.1085/jgp.201210789.
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Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.
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Samuilova, Olga, Camilla Krogerus, Igor Fabrichniy, and Timo Hyypiä. "ATP Hydrolysis and AMP Kinase Activities of Nonstructural Protein 2C of Human Parechovirus 1." Journal of Virology 80, no. 2 (January 15, 2006): 1053–58. http://dx.doi.org/10.1128/jvi.80.2.1053-1058.2006.
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ABSTRACT The highly conserved picornavirus 2C proteins, thought to be involved in genome replication, contain three motifs found in NTPases/helicases of superfamily III. We report that human parechovirus 1 2C displays Mg2+-dependent ATP diphosphohydrolase activity in vitro, whereas other nucleoside triphosphates are not substrates for the hydrolysis. We also found that the 2C protein has an enzymatic activity that converts AMP to a corresponding diphosphate using ADP or ATP as a phosphate donor. In addition, we observed that ATP hydrolysis results in 2C autophosphorylation. These findings indicate that the parechovirus 2C protein has enzymatic activities, which may contribute to several functions in the viral replication cycle.
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Horscroft, Nigel J., and Polly Roy. "NTP binding and phosphohydrolase activity associated with purified bluetongue virus non-structural protein NS2." Journal of General Virology 81, no. 8 (August 1, 2000): 1961–65. http://dx.doi.org/10.1099/0022-1317-81-8-1961.
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The bluetongue virus ssRNA-binding protein, NS2, is a phosphoprotein that forms viral inclusion bodies in infected cells. Recombinant NS2 was expressed in the baculovirus expression system and purified to homogeneity from insect cells. Purified NS2 bound nucleosides. Further investigation revealed that the protein bound ATP and GTP and could hydrolyse both nucleosides to their corresponding NMPs, with a higher efficiency for the hydrolysis of ATP. The increased efficiency of hydrolysis of ATP correlated with a higher binding affinity of NS2 for ATP than GTP. Ca2+, Mg2+ and Mn2+ were able to function as the required divalent cation in the reactions. The phosphohydrolase activity was not sensitive to ouabain, an inhibitor of cellular ATPases, suggesting that this activity was not the result of a cellular contaminant.
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Gômez-Puyou, M. Tuena de, Orlando B. Martins, and A. Gômez-Puyou. "Synthesis and hydrolysis of ATP by the mitochondrial ATP synthase." Biochemistry and Cell Biology 66, no. 7 (July 1, 1988): 677–82. http://dx.doi.org/10.1139/o88-077.
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A brief summary of the factors that control synthesis and hydrolysis of ATP by the mitochondrial H+-ATP synthase is made. Particular emphasis is placed on the role of the natural ATPase inhibitor protein. It is clear from the existing data obtained with a number of agents that there is no correlation between variations of the rate of ATP hydrolysis and ATP synthesis as driven by respiration. The mechanism by which each condition differentially affects the two activities is not entirely known. For the case of the natural ATPase inhibitor protein, it appears that the protein controls the kinetics of the enzyme. This control seems essential for achieving maximal accumulation of ATP during electron transport in systems that contain relatively high concentrations of ATP.
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Knight, Alex E., and Justin E. Molloy. "Coupling ATP hydrolysis to mechanical work." Nature Cell Biology 1, no. 4 (August 1999): E87—E89. http://dx.doi.org/10.1038/12083.
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Wittinghofer, Alfred. "GTP and ATP hydrolysis in biology." Biopolymers 105, no. 8 (May 20, 2016): 419–21. http://dx.doi.org/10.1002/bip.22867.
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CHALOVICH, JOSEPH M., MARK E. HEMRIC, and LALY VELAZ. "Regulation of ATP Hydrolysis by Caldesmon." Annals of the New York Academy of Sciences 599, no. 1 Cell Lineages (August 1990): 85–99. http://dx.doi.org/10.1111/j.1749-6632.1990.tb42367.x.
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Strotmann, Heinrich, Sigrid Kleefeld, and Detlev Lohse. "Control of ATP hydrolysis in chloroplasts." FEBS Letters 221, no. 2 (September 14, 1987): 265–69. http://dx.doi.org/10.1016/0014-5793(87)80938-9.
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Sauna, Zuben E., and Suresh V. Ambudkar. "Characterization of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein." Journal of Biological Chemistry 276, no. 15 (January 11, 2001): 11653–61. http://dx.doi.org/10.1074/jbc.m011294200.
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P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis à visthe substrate, they show comparablet12for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.
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Keller, David, Seema Singh, Paola Turina, Roderick Capaldi, and Carlos Bustamante. "Structure of ATP synthase by SFM and single-particle image analysis." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 722–23. http://dx.doi.org/10.1017/s0424820100139986.
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F1Fo ATP synthases are the proteins responsible for the synthesis of ATP in oxidative phosphorylation, and are present in some form in all aerobic organisms, both prokaryotic and eukaryotic. They use the energy stored in a transmembrane proton gradient (which is generated by other members of the oxidative phosphorylation pathway) to synthesize ATP from ADP and Pi or, working in reverse, to pump protons across the membrane using the energy of ATP hydrolysis. The full protein has two sectors, F1 and Fo. F1 is normally bound to Fo (which is membrane integrated), but is water soluble when dissociated. The F1 sector contains the sites which bind ADP and catalyze its conversion to ATP. The Fo sector contains a channel which allows protons to to cross the membrane, dissipating the transmembrane chemical potential. By an unknown mechanism this translocation of protons through Fo is coupled to the hydrolysis or synthesis of ATP in F1, so that the energy released in hydrolysis of ATP can drive the motion of protons against an electrochemical potential, or the energy of translocating protons can be used to form high energy ADP-Pi bonds.
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Weinreich, Frank, John R. Riordan, and Georg Nagel. "Dual Effects of Adp and Adenylylimidodiphosphate on Cftr Channel Kinetics Show Binding to Two Different Nucleotide Binding Sites." Journal of General Physiology 114, no. 1 (July 1, 1999): 55–70. http://dx.doi.org/10.1085/jgp.114.1.55.
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The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.
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BISWAS, Indranil, and Ravi VIJAYVARGIA. "Heteroduplex DNA and ATP induced conformational changes of a MutS mismatch repair protein from Thermus aquaticus." Biochemical Journal 347, no. 3 (April 25, 2000): 881–86. http://dx.doi.org/10.1042/bj3470881.
Full textAbstract:
ATP hydrolysis by MutS homologues is required for the function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is not very clear. We have examined the role of ATP hydrolysis in oligomerization of Thermus aquaticus (Taq) MutS protein in solution. Analytical gel filtration and cross-linking of MutS protein with disuccinimidyl suburate suggest that TaqMutS is a dimer in the presence of ATP. ATP binding and hydrolysis by TaqMutS reduces the heteroduplex-DNA binding by the protein. Using limited proteolysis we detected extensive conformational changes of the TaqMutS protein in the presence of ATP and heteroduplex DNA. Heteroduplex-DNA binding is necessary for the observed conformational changes since F39A mutant protein defective in DNA binding does not display ATP-induced conformational changes. The implications of the observed conformational changes in the MutS protein are discussed with respect to two different models proposed for the role of ATP hydrolysis by MutS in DNA mismatch repair.
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Reddy, M. M., and P. M. Quinton. "Hydrolytic and nonhydrolytic interactions in the ATP regulation of CFTR Cl- conductance." American Journal of Physiology-Cell Physiology 271, no. 1 (July 1, 1996): C35—C42. http://dx.doi.org/10.1152/ajpcell.1996.271.1.c35.
Full textAbstract:
Previously, we showed in the native sweat duct that, in the presence of 0.1-0.5 mM ATP, nonhydrolyzable ATP analogue adenosine 5'-adenylylimidodiphosphate (AMP-PNP) can activate cystic fibrosis transmembrane conductance regulator Cl- conductance (CFTR GCl) (15). The objective of this study is to determine if 1) nonhydrolytic ATP binding alone can activate CFTR GCl after stable phosphorylation [in the presence of adenosine 5'-O-(3-thiotriphosphate) and phosphatase inhibition cocktail] of CFTR or 2) an ATP hydrolysis (in addition to phosphorylation) is required to support subsequent nonhydrolytic ATP regulation of CFTR GCl. We show that stably phosphorylated CFTR could only be activated by AMP-PNP in the presence of a small background ATP concentration. However, AMP-PNP can sustain previously activated CFTR GCl in the absence of ATP, even though Mg2+ is required for phosphorylation activation of CFTR GCl. However, once stably phosphorylated, ATP activation of CFTR GCl is independent of Mg2+. Our results show that both hydrolytic and nonhydrolytic interactions regulate CFTR GCl in vivo. Nonhydrolytic ATP interaction plays a significant role in both activation and deactivation of CFTR GCl.