- Analytical Techniques for the Determination of Chemical Exchange Rate Constants with Application to the Creatine Kinase Reaction
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A method for determining biochemical exchange rates for analysis of inversion recovery experiments is described.As used in the current application, the technique involves fitting multiexponential equations to the magnetization recovery curves for PCr and ATP.From the parameters of the exponential fits expressions for the forward and reverse rate constants are derived.Results obtained with this technique were compared with inversion transfer and with previously published results using saturation transfer.The addition of a third exchanging species was also investigated and found to have no significant effect on the calculated values for the forward and reverse rate constants.In addition, the feasibility of using an abbreviated 6-point sampling strategy was evaluated; values for the rate constants were similar to those obtained using all 21 data points.The results of this study indicate that chemical exchange rate constants can be determined using inversion recovery techniques which avoid many of the difficulties associated with selective excitation methods.
- Sorce, Dennis J.,Sciacca, Robert R.,Keller, Andrew M.
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- Practical Enzymatic Synthesis of Adenosine 5'-O-(3-Thiotriphosphate) (ATP-γ-S)
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An enzymatic procedure for the synthesis of adenosine 5'-O-(3-thiotriphosphate) (ATP-γ-S) on a 50-mmol scale from dihydroxyacetone, sodium thiophosphate, ADP, and phosphoenol pyruvate is described.The synthesis uses polyacrylamide gel immobilized glycerokinase coupled to a pyruvate kinase catalyzed ATP cofactor regeneration system, and polyacrylamide gel immobilized triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, and phosphoglycerate kinase coupled to a lactate dehydrogenase catalyzed NAD cofactor regeneration system.The ATP-γ-S is purified by adsorption on Dowex 1 and isolated as the sodium or barium salts in ca. 90 percent purity.
- Abril, Obsidiana,Crans, Debbie C.,Whitesides, George M.
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- Graphene oxide enhanced specificity at aptamer and its application to multiplexed enzymatic activity sensing
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We explore the effect of sufficient GO on the property of a dye labeled adenosine 5′-triphosphate (ATP) aptamer (P) which shows similar affinity and specificity for ATP and its analogues including adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), and adenosine (AD). It is found that ATP and its analogues give rise to fluorescence recovery of GO-quenched P to a different extent (in the order of ATP > AD > ADP > AMP), and the difference becomes larger when increasing the concentration of GO in a certain range, implying an improvement of specificity of the ATP aptamer. Based on this finding, a fluorescence turn-on assay for alkaline phosphatase (ALP) and creatine kinase (CK) is proposed, by using AMP and ADP as the substrate, respectively. Specifically, the GO-quenched P system containing substrate shows low fluorescence intensity. In the presence of target enzyme, the substrate is converted into either AD or ATP which have higher affinity with P, resulting in stronger fluorescence of the mixture of P and GO. The entire assay is sensitive and selective. More importantly, the ability of GO with suitable concentration to improve the specificity of aptamers not only offers an exciting new way to detect protease, but also is valuable for developing the application of GO and aptamers in the biosensing field and is expected to be used in aptamer screening systems, to improve the specificity of screened aptamers.
- Xing, Xiaojing,Liu, Xueguo,Zhou, Ying,Xu, Dangdang,Pang, Daiwen,Tang, Hongwu
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- Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity
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Synthesis of adenine derivative of triphosphono-γ -(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-γ -(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.
- Moosavi-Movahedi, Ali A.,Hakimelahi, Shahram,Chamani, Jamshid,Khodarahmi, Ghadam Ali,Hassanzadeh, Farshid,Luo, Fen-Tair,Ly, Tai Wei,Shia, Kak-Shan,Yen, Chi-Feng,Jain, Moti L.,Kulatheeswaran, Ramasamy,Xue, Cuihua,Pasdar, Manijeh,Hakimelahi, Gholam Hossein
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- Conformational dynamics and allostery in pyruvate kinase
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Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1, 6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1, 6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit This destabilizes the βstrands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1, 6-bisphosphate affects the active site.
- Donovan, Katherine A.,Zhu, Shaolong,Liuni, Peter,Peng, Fen,Kessans, Sarah A.,Wilson, Derek J.,Dobson, Renwick C.J.
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- Formation of ATP by photochemical excitation of benzoquinones in dimethylacetamide solution
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A new method of adenosine triphosphate production is described which involves photo-excitation of p-benzoquinones under the presence of adenosine diphosphate and inorganic phosphate in N,N-dimethylacetamide solution. A possible mechanism for the reaction is presented.
- Lee, Hung-Wen,Huang, Kunpo
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- Physiological and biochemical characterization of three nucleoside diphosphate kinase isozymes from rice (Oryza sativa L.)
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Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. In this study, we examined the subcellular localization, tissue-specific gene expression, and enzymatic characteristics of three rice NDPK isozymes (OsNDPK1-OsNDPK3). Sequence comparison of the three OsNDPKs suggested differential subcellular localization. Transient expression of green fluorescence protein-fused proteins in onion cells indicated that OsNDPK2 and OsNDPK3 are localized to plastid and mitochondria respectively, while OsNDPK1 is localized to the cytosol. Expression analysis indicated that all the OsNDPKs are expressed in the leaf, leaf sheath, and immature seeds, except for OsNDPK1, in the leaf sheath. Recombinant OsNDPK2 and OsNDPK3 showed lower optimum pH and higher stability under acidic pH than OsNDPK1. In ATP formation, all the OsNDPKs displayed lower Km values for the second substrate, ADP, than for the first substrate, NTP, and showed lowest and highest K m values for GTP and CTP respectively.
- Kihara, Akihiko,Saburi, Wataru,Wakuta, Shinji,Kim, Myung-Hee,Hamada, Shigeki,Ito, Hiroyuki,Imai, Ryozo,Matsui, Hirokazu
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- Biomimetic One-Pot Synthesis of Nucleotide Phosphates
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A strongly hydrophobic rigid diammonium, 1, an efficient extracting and transporting phase transfer reagent with high specificity for the pyrophosphate grouping, was used to synthesize ADP, ATP or ADP-NH2 in a hydrophobic medium.Thus, practically pure ADP-NH2 was obtained in 65 percent yield within 2 min.
- Tabushi, Iwao,Imuta, Jun-ichi
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- Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant staphylococcus aureus
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Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose 5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4) the kinetic efficiency (kcat/S0.5) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state, toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the sequence containing the CT domain (Gly473-Leu585) plays a substantial role in enzyme activity and comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP (e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and mode of allosteric regulation which may aid in the development of clinically important drugs targeting this enzyme and further define the role of the extra C-terminal domain in modulating the enzyme's activity.
- Zoraghi, Roya,See, Raymond H.,Gong, Huansheng,Lian, Tian,Swayze, Rick,Finlay, B. Brett,Brunham, Robert C.,McMaster, William R.,Reiner, Neil E.
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- STOICHIOMETRY OF PROTON TRANSLOCATION DURING PHOTOSYNTHESIS.
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Formation of ATP in photosynthesis takes place at the expense of free energy stored in the gradient of protons across the thylakoid membrane. Set up of the proton gradient is accomplished by the light-driven electron transport reactions. The stoichiometry of H** plus translocation across the thylakoid membrane is determined with respect to electron flow and ATP synthesis. Direct information is obtained on the basis of the H** plus flux measurement. Depending on the experimental conditions two or three H** plus are translocated from outside into the aqueous inner phase of the thylakoid vesicles for each electron which is transfered through the electron transport chain. Three H** plus are translocated from inside to outside across the ATPase for each ATP molecule which is synthesized. Mechanistic and energetic consequences are discussed.
- Rathenow,Rumberg
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- Supramolecularly Assembled Nanocomposites as Biomimetic Chloroplasts for Enhancement of Photophosphorylation
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Prototypes of natural biosystems provide opportunities for artificial biomimetic systems to break the limits of natural reactions and achieve output control. However, mimicking unique natural structures and ingenious functions remains a challenge. Now, multiple biochemical reactions were integrated into artificially designed compartments via molecular assembly. First, multicompartmental silica nanoparticles with hierarchical structures that mimic the chloroplasts were obtained by a templated synthesis. Then, photoacid generators and ATPase-liposomes were assembled inside and outside of silica compartments, respectively. Upon light illumination, protons produced by a photoacid generator in the confined space can drive the liposome-embedded enzyme ATPase towards ATP synthesis, which mimics the photophosphorylation process in vitro. The method enables fabrication of bioinspired nanoreactors for photobiocatalysis and provides insight for understanding sophisticated biochemical reactions.
- Li, Yue,Feng, Xiyun,Wang, Anhe,Yang, Yang,Fei, Jinbo,Sun, Bingbing,Jia, Yi,Li, Junbai
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- Metabolic pathways for activation of the antiviral agent 9-(2- phosphonylmethoxyethyl)adenine in human lymphoid cells
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9-(2-Phosphonylmethoxyethyl)adenine (PMEA), the acyclic phosphonate analog of adenine monophosphate, is a promising antiviral drug with activity against herpesviruses, Epstein-Barr virus, and retroviruses, including the human immunodeficiency virus. In order to be active, it must be converted to the diphosphate derivative, the putative inhibitor of viral DNA polymerases. The metabolic pathway responsible for activation of PMEA is unclear. The metabolism of PMEA was investigated in human T-lymphoid cells (CEMss) and a PMEA-resistant subline (CEMss(r-1)) with a partial deficiency in adenylate kinase activity. Experiments with [3H]PMEA showed that extracts of CEMss phosphorylated PMEA to its mono- and diphosphate in the presence of ATP as the phosphate donor. No other nucleotides or 5-phosphoribosyl pyrophosphate displayed appreciable activity as a phosphate donor. Subcellular fractionation experiments showed that CEMss cells contained two nucleotide kinase activities, one in mitochondria and one in the cytosol, which phosphorylated PMEA. The PMEA-resistant CEMss mutant proved to have a deficiency in the mitochondrial adenylate kinase activity, indicating that this enzyme was important in the phosphorylation of PMEA. Other effective antiviral purine phosphonate derivatives of PMEA showed a profile of phosphorylating activity similar to that of PMEA. By comparison, phosphorylation of the pyrimidine analog (S)-1-(3-hydroxy-2- phosphonylmethoxypropyl) cytosine proceeded by an enzyme present in the cytosol. We conclude from these studies that adenylate kinase which has been localized in the intermembrane space of mitochondria is the major route for PMEA phosphorylation in CEMss cells but that another hitherto unidentified enzyme(s) present in the cytosol may contribute to the anabolism of the phosphonates.
- Robbins,Greenhaw,Connelly,Fridland
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- Fe(III)-ion-Catalysed Non-enzymatic Transformation of ADP into ATP
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Non-enzymatic phosphorylation of ADP to ATP has been found to occur readily when Fe(III) ion is present in aqueous solutions of ADP and acetyl phosphate.As long as the reaction temperature is low enough (25 deg C or less), the phosphorylation of ADP to ATP proceeds to about 20percent conversion with 100percent selectivity.At higher temperatures, the yield decreases owing to the hydrolysis of acetyl phosphate.Among several phosphoryl donors studied, acetyl phosphate was found to be the best phosphorylating reagent.Creatine phosphate also produced ATP but the efficiency was very low.The phosphorylation of AMP was unsuccessful, even with acetyl phosphate.
- Kitani, Akira,Tsunetsugu, Shu-ichi,Sasaki, Kazuo
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- Spatiotemporal effects of a bioautocatalytic chemical wave revealed by time-resolved mass spectrometry
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Mass spectrometry has been implemented as an on-line detection tool to monitor transmission of chemical signals due to natural processes such as diffusion and convection as well as a bienzymatic autocatalytic process. It was found that the enzyme-accelerated chemical wave propagates faster than the chemical wave propelled by other processes. The two enzymes (pyruvate kinase and adenylate kinase), involved in the process, work co-operatively catalysing production of adenosine diphosphate (ADP) and adenosine triphosphate (ATP), and induce formation of a front propagating along a high-aspect-ratio drift cell towards the ion source of an ion trap mass spectrometer. Isotopically labelled 13C-ATP was used as the trigger of the accelerated chemical wave. Using this substrate, one could easily distinguish between the two chemical waves (passive and accelerated) in a single experiment, reducing the bias due to the inherent experimental instabilities. We think that-following further improvements-the process described in this report may find applications in bioengineered systems, in which chemical signals need to be transmitted over macroscopic distances.
- Ting, Hsu,Urban, Pawel L.
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- Boric Acid-Fueled ATP Synthesis by FoF1 ATP Synthase Reconstituted in a Supramolecular Architecture
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Significant strides toward producing biochemical fuels have been achieved by mimicking natural oxidative and photosynthetic phosphorylation. Here, different from these strategies, we explore boric acid as a fuel for tuneable synthesis of energy-storing molecules in a cell-like supramolecular architecture. Specifically, a proton locked in boric acid is released in a modulated fashion by the choice of polyols. As a consequence, controlled proton gradients across the lipid membrane are established to drive ATP synthase embedded in the biomimetic architecture, which facilitates tuneable ATP production. This strategy paves a unique route to achieve highly efficient bioenergy conversion, holding broad applications in synthesis and devices that require biochemical fuels.
- Xu, Xia,Fei, Jinbo,Xu, Youqian,Li, Guangle,Dong, Weiguang,Xue, Huimin,Li, Junbai
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- Carbamate transport in carbamoyl phosphate synthetase: A theoretical and experimental investigation
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The transport of carbamate through the large subunit of carbamoyl phosphate synthetase (CPS) from Escherichia coli was investigated by molecular dynamics and site-directed mutagenesis. Carbamate, the product of the reaction involving ATP, bicarbonate, and ammonia, must be delivered from the site of formation to the site of utilization by traveling nearly 40 A within the enzyme. Potentials of mean force (PMF) calculations along the entire tunnel for the translocation of carbamate indicate that the tunnel is composed of three continuous water pockets and two narrow connecting parts, near Ala-23 and Gly-575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets were filled with about 21, 9, and 9 waters, respectively, and the corresponding relative free energies of carbamate residing in these free energy minima are 5.8, 0, and 1.6 kcal/mol, respectively. The release of phosphate into solution at the site for the formation of carbamate allows the side chain of Arg-306 to rotate toward Glu-25, Glu-383, and Glu-604. This rotation is virtually prohibited by a barrier of at least 23 kcal/mol when phosphate remains bound. This conformational change not only opens the entrance of the tunnel but also shields the charge charge repulsion from the three glutamate residues when carbamate passes through the tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. The mutants retained only 1.7% and 3.8% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS, respectively.
- Lund, Liliya,Fan, Yubo,Shao, Qiang,Gao, Yi Qin,Raushel, Frank M.
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- Stereospecificity, substrate, and inhibitory properties of nucleoside diphosphate analogs for creatine and pyruvate kinases
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Antiviral α-P-borano substituted NTPs are promising chain terminators targeting HIV reverse transcriptase (RT). Activation of antiviral nucleoside diphosphates (NDPs) to NTPs may be carried out by pyruvate kinase (PK) and creatine kinase (CK). Herein, are presented the effects of nucleobase, ribose, and α-phosphate substitutions on substrate specificities of CK and PK. Both enzymes showed two binding modes and negative cooperativity with respect to substrate binding. The stereospecificity and inhibition of ADP phosphorylation by α-P-borano substituted NDP (NDPαB) stereoisomers were also investigated. The Sp-ADPαB isomer was a 70-fold better substrate for CK than the Rp isomer, whereas PK preferred the Rp isomer of NDPαBs. For CK, the Sp-ADPαB isomer was a competitive inhibitor; for PK, the Rp-ADPαB isomer was a poor competitive inhibitor and the Sp-ADPαB isomer was a poor non-competitive inhibitor. Taken together, these data suggest that, although the Rp-NDPαB isomer would be minimally phosphorylated by CK or PK, it should not inhibit either enzyme.
- Wennefors, Charlotta K.,Dobrikov, Mikhail I.,Xu, Zhihong,Li, Ping,Shaw, Barbara Ramsay
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- Class III Polyphosphate Kinase 2 Enzymes Catalyze the Pyrophosphorylation of Adenosine-5′-Monophosphate
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Polyphosphate kinase 2 (PPK2) transfer phosphate from inorganic polyphosphate to nucleotides. According to their activity, PPK2 enzymes are classified into three groups. Among them, class III enzymes catalyze both the phosphorylation of nucleotide mono- to diphosphates and di- to triphosphates by using polyphosphate, which is a very inexpensive substrate. Therefore, class III enzymes are very attractive for use in biotechnological applications. Despite several studies on class III enzymes, a detailed mechanism of how phosphate is transferred from the polyphosphate to the nucleotide remains to be elucidated. Herein, it is reported that PPK2 class III enzymes from two different bacterial species catalyze the phosphorylation of adenosine mono- (AMP) into triphosphate (ATP) not only through step-by-step phosphorylation, but also by pyrophosphorylation. These are the first PPK2 enzymes that have been shown to possess polyphosphate-dependent pyrophosphorylation activity.
- Ogawa, Marin,Uyeda, Atsuko,Harada, Kazuo,Sato, Yu,Kato, Yasuhiko,Watanabe, Hajime,Honda, Kohsuke,Matsuura, Tomoaki
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- ATP regeneration by a single polyphosphate kinase powers multigram-scale aldehyde synthesisin vitro
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ATP recycling systems are required to avoid the addition of stoichiometric quantities of cofactor and facilitate industrial implementation of ATP-dependent enzymes. One factor that limits the biocatalytic application of these enzymes is the lack of a scalable AMP to ATP regeneration system. Whole-cells or a combination of purified enzymes are often exploited for ATP regeneration from AMP, whereas cell free systems comprising a single crude enzyme preparation would be preferred. To establish such a system, we focussed on polyphosphate kinases (PPKs) to find a single enzyme that could be used to power ATP-consuming reactions. Screening of some previously reported PPKs revealed limitations of these biocatalysts for scale-up purposes. As such, a panel of novel putative PPK2-III enzymes was constructed and compared to characterised enzymes belonging to the same class. Multidimensional small-scale screening revealed that PPK12 (from an unclassifiedErysipelotrichaceaebacterium) displays enhanced expression levels, ATP formation rates, polyphosphate tolerance and stability under a variety of harsh conditions. The carboxylic acid reductase (CAR) catalysed reduction of carboxylates to aldehydes was chosen as a model reaction to test the applicability of PPK12 as a bifunctional biocatalyst for ATP regeneration from AMP. The implementation of the identified ATP-recycling enzyme provided the first example of cell free multigram-scale aldehyde synthesis employing enzymes and a single PPK2-III, paving the way for affordable scalable ATP regeneration technologies.
- Tavanti, Michele,Hosford, Joseph,Lloyd, Richard C.,Brown, Murray J. B.
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supporting information
p. 828 - 837
(2021/02/09)
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- ATP Regeneration System in Chemoenzymatic Amide Bond Formation with Thermophilic CoA Ligase
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CoA ligases are enzymes catalyzing the ATP-dependent addition of coenzyme A to carboxylic acids in two steps through an adenylate intermediate. This intermediate can be diverted by a nucleophilic non enzymatic addition of amine to get the corresponding amide for synthetic purposes. To this end, we selected thermophilic CoA ligases to study the conversion of various carboxylic acids into their amide counterparts. To limit the use of ATP, we implemented an ATP regeneration system combining polyphosphate kinase 2 (PPK2 Class III) and inorganic pyrophosphatase. Suitability of this system was illustrated by the lab-scale chemoenzymatic synthesis of N-methylbutyrylamide in 77 % yield using low enzyme loading and 5 % molar ATP.
- Lelièvre, Chloé M.,Balandras, Mélanie,Petit, Jean-Louis,Vergne-Vaxelaire, Carine,Zaparucha, Anne
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p. 1184 - 1189
(2020/01/22)
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- Photo-electrochemical Bioanalysis of Guanosine Monophosphate Using Coupled Enzymatic Reactions at a CdS/ZnS Quantum Dot Electrode
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A photo-electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)-dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co-substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co-substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD+. This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.
- Sabir, Nadeem,Khan, Nazimuddin,V?lkner, Johannes,Widdascheck, Felix,Del Pino, Pablo,Witte, Gregor,Riedel, Marc,Lisdat, Fred,Konrad, Manfred,Parak, Wolfgang J.
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p. 5844 - 5850
(2016/01/25)
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- A cross-chiral RNA polymerase ribozyme
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Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer. This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition or chiral symmetry was broken through chemical processes before the origin of RNA-based life. Once an RNA enzyme arose that could catalyse the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating d- and l-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyse the templated polymerization of activated mononucleotides of the opposite handedness. Here we develop such a cross-chiral RNA polymerase, using in vitro evolution starting from a population of random-sequence RNAs. The d-RNA enzyme, consisting of 83 nucleotides, catalyses the joining of l-mono- or oligonucleotide substrates on a complementary l-RNA template, and similar behaviour occurs for the l-enzyme with d-substrates and a dlate. Chiral inhibition is avoided because the 10 6 -fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme € s activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides.
- Sczepanski, Jonathan T.,Joyce, Gerald F.
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p. 440 - 442
(2015/02/19)
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- Dual-color control of nucleotide polymerization sensed by a fluorescence actuator
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Spatial and temporal control of molecular mechanisms can be achieved using photolabile bonds that connect biomolecules to protective caging groups, which can be cleaved upon irradiation of a specific wavelength, releasing the biomolecule ready-to-use. Here we apply and improve a previously reported strategy to tightly control in vitro transcription reactions. The strategy involves two caging molecules that block both ATP and GTP nucleotides. Additionally, we designed a molecular beacon complementary to the synthesized mRNA to infer its presence through a light signal. Upon release of both nucleotides through a specific monochromatic light (390 and 325 nm) we attain a light signal indicative of a successful in vitro transcription reaction. Similarly, in the absence of irradiation, no intense fluorescence signal was obtained. We believe this strategy could further be applied to DNA synthesis or the development of logic gates. This journal is
- Reimao-Pinto, Madalena M.,Cordeiro, Ana,Almeida, Carina,Pinheiro, Andre V.,Moro, Artur,Lima, Joao C.,Baptista, Pedro V.
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p. 751 - 756
(2014/05/06)
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- Molecular characterization and mutational analysis of recombinant diadenosine 5′,5″-P1,P4-tetraphosphate hydrolase from Plasmodium falciparum
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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.
- Osman, Waleed,Endo, Satoshi,Oh-hashi, Kentaro,Kitamura, Yoshiaki,Kitade, Yukio
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experimental part
p. 1191 - 1196
(2012/08/07)
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- ATP selective acridone based fluorescent probes for monitoring of metabolic events
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Acridones carrying an appropriate substituent at N-10 showed significant fluorescence changes on interacting with ATP in HEPES buffer at pH 7.2. The selectivity and sufficient binding of these probes with ATP could be useful for monitoring of metabolic processes.
- Kaur, Jatinder,Singh, Palwinder
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scheme or table
p. 4472 - 4474
(2011/07/07)
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- Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: Implications for the design of better anti-HIV analogues
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The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the d-/l-configurations of the nucleotides, the l-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the l-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an l-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.
- Varga, Andrea,Chaloin, Laurent,Sagi, Gyula,Sendula, Robert,Graczer, Eva,Liliom, Karoly,Zavodszky, Peter,Lionne, Corinne,Vas, Maria
-
experimental part
p. 1863 - 1873
(2012/04/17)
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- An improved one-pot synthesis of nucleoside 5'-triphosphate analogues
-
Nucleoside 5'-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5'-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to -15°C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using 31P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.
- Gillerman, Irina,Fischer, Bilha
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p. 245 - 256
(2011/08/06)
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- Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [α-32P]d/rNTP
-
Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d2CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, k m and k cat were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [α- 32P]rNTP and [α-32P]dNTP.
- Skoblov,Mikoulinskaia,Taran,Miroshnikov,Feofanov,Skoblov
-
experimental part
p. 734 - 738
(2010/08/07)
-
- A kinetic study of the rat liver adenosine kinase reverse reaction
-
Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH3. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels. Copyright Taylor & Francis Group, LLC.
- Vannoni,Giglioni,Santoro,Aceto,Marinello,Leoncini
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p. 872 - 875
(2008/12/21)
-
- Selective diphosphorylation, dithiodiphosphorylation, triphosphorylation, and trithiotriphosphorylation of unprotected carbohydrates and nucleosides
-
(Chemical Equation Presented) Aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with diphosphitylating and triphosphitylating reagents to yield the corresponding polymer-bound diphosphitylating and triphosphitylating reagents, respectively. A number of unprotected carbohydrates and nucleosides were reacted with the polymer-bound reagents. Oxidation with tert-butyl hydroperoxide or sulfurization with Beaucage's reagent, followed by removal of cyanoethoxy group with DBU and the acidic cleavage, respectively, afforded only one type of monosubstituted nucleoside and carbohydrate diphosphates, dithiodiphosphates, triphosphates, and trithiotriphosphates with high regioselectivity.
- Ahmadibeni, Yousef,Parang, Keykavous
-
p. 5589 - 5592
(2007/10/03)
-
- Synthesis of nucleoside α-thiotriphosphates via an oxathiaphospholane approach
-
(Chemical Equation Presented) Nucleoside 5′-O-(α- thiotriphosphates) were obtained in reactions of the appropriate nucleoside 5′-O-(2-thio-1,3,2-oxathiaphospholanes) with pyrophosphate in the presence of DBU. The presented method allows also for preparation of α-seleno congeners and corresponding α-modified diphosphates.
- Misiura, Konrad,Szymanowicz, Daria,Stec, Wojciech J.
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p. 2217 - 2220
(2007/10/03)
-
- Hydrolytic reactions of diadenosine 5′,5′-triphosphate
-
The hydrolysis of diadenosine 5′,5′-triphosphate to AMP and ADP has been studied over a wide pH-range. Under acidic conditions the reaction shows a first-order dependence on the hydronium ion concentration. Below pH 3 the rate-increase begins to level off. From pH 6 to 9 the hydrolysis is slow and pH-independent. Base-catalysed hydrolysis is observed in NaOH-solutions. Under alkaline conditions an intramolecular nucleophilic attack on the phosphate producing 3′,5′-cAMP is also observed, but it is slower than the intermolecular reaction. Depurination of the adenosine moieties competes with the hydrolysis both under acidic and alkaline conditions, but the mechanisms are different. The temperature-dependence of the hydrolysis of Ap3A and the depurination of adenosine moieties were studied under acidic conditions, and the activation parameters of the reactions were calculated. The results of the work reflect the fact that the negatively charged polyphosphate group is very resistant towards nucleophilic attack. An efficient catalysis is only observed under acidic conditions, where the phosphate group becomes protonated. General acids or bases did not catalyse the hydrolysis. Furthermore, hydroxide ion catalysed cleavage is only observed at high base concentrations and other negatively charged nucleophiles did not attack the phosphate groups of diadenosine polyphosphates.
- Mikkola, Satu
-
p. 770 - 776
(2007/10/03)
-
- Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs
-
Nucleoside analogs used in antiviral therapies need to be phosphorylated to their tri-phospho counterparts in order to be active on their cellular target. Human phosphoglycerate kinase (hPGK) was recently reported to participate in the last step of phosphorylation of cytidine l-nucleotide derivatives [Krishnan PGE, Lam W, Dutschman GE, Grill SP, Cheng YC. Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of l-nucleoside analogs, a new class of anticancer and antiviral agents. J Biol Chem 2003;278:36726-32]. In the present work, we extended the enzymatic study of human PGK specificity to purine and pyrimidine nucleotide derivatives in both d- and l-configuration. Human PGK demonstrated catalytic efficiencies in the 104-10 5 M-1 s-1 range for purine ribo-, deoxyribo- and dideoxyribonucleotide derivatives, either in d- or l-configuration. In contrast, it was poorly active with natural pyrimidine d-nucleotides (less than 103 M-1 s-1). Pyrimidine l-enantiomers, which are promising therapeutic analogs against B hepatitis, were 2-25 times better substrates than their d-counterparts. The broad specificity of substrate of human PGK suggests that this enzyme may be involved in the cellular activation of several antiviral nucleoside analogs including dideoxyinosine, acyclovir, l-2′-deoxycytosine and l-2′-deoxythymidine.
- Gallois-Montbrun, Sarah,Faraj, Abdesslem,Seclaman, Edward,Sommadossi, Jean-Pierre,Deville-Bonne, Dominique,Véron, Michel
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p. 1749 - 1756
(2007/10/03)
-
- Photochemical Reaction Mechanisms of 2-Nitrobenzyl Compounds: Methyl Ethers and Caged ATP
-
The mechanism of methanol photorelease from 2-nitrobenzyl methyl ether (1) and 1-(2-nitrophenyl)ethyl methyl ether (2), and of ATP release from adenosine-5′-triphosphate-[P3-(1-(2-nitrophenyl)-ethyl)] ester ('caged ATP', 3) was studied in various solvents by laser flash photolysis with UV-vis and IR detection. In addition to the well-known primary aci-nitro transients (A, λmax ≈ 400 nm), two further intermediates preceding the release of methanol, namely the corresponding 1,3-dihydrobenz[c]isoxazol-1-ol derivatives (B) and 2-nitrosobenzyl hemiacetals (C), were identified. The dependencies of the reaction rates of A-C on pH and buffer concentrations in aqueous solution were studied in detail. Substantial revision of previously proposed reaction mechanisms for substrate release from 2-nitrobenzyl protecting groups is required: (a) A novel reaction pathway of the aci-tautomers A prevailing in buffered aqueous solutions, e.g., phosphate buffer with pH 7, was found. (b) The cyclic intermediates B were identified for the first time as the products formed by the decay of the aci-tautomers A in solution. A recently proposed reaction pathway bypassing intermediates B (Corrie et al. J. Am. Chem. Soc., 2003, 125, 8546-8554) is shown not to be operative. (c) Hemiacetals C limit the release rate of both 1 (pH 8) and 2 (pH 10). This observation is in contrast to a recent claim for related 2-nitrobenzyl methyl ethers (Corrie et al.). Our findings are important for potential applications of the 2-nitrobenzyl protecting group in the determination of physiological response times to bioagents ('caged compounds').
- Il'ichev, Yuri V.,Schwoerer, Markus A.,Wirz, Jakob
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p. 4581 - 4595
(2007/10/03)
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- Thermodynamics of the hydrolysis reactions of adenosine 3′,5′-(cyclic)phosphate(aq) and phosphoenolpyruvate(aq); the standard molar formation properties of 3′,5′-(cyclic)phosphate(aq) and phosphoenolpyruvate(aq)
-
Molar calorimetric enthalpy changes ΔrHm(cal) have been measured for the biochemical reactions {cAMP(aq) + H2O(l) = AMP(aq)} and {PEP(aq) + H2O(l) = pyruvate(aq) + phosphate(aq)}. The reactions were catalyzed, respectively, by phosphodiesterase 3prime;,5prime;-cyclic nucleotide and by alkaline phosphatase. The results were analyzed by using a chemical equilibrium model to obtain values of standard molar enthalpies of reaction ΔrHmo for the respective reference reactions {cAMp-(aq) + H2O(l) = HAMP-(aq)} and {PEP3-(aq) + H2O(l) = pyruvate-(aq) + HPO42-(aq)}. Literature values of the apparent equilibrium constants K′ for the reactions {ATP(aq) = cAMP(aq) + pyrophosphate(aq)K {ATP(aq) + pyruvate(aq) = ADP(aq) + PEP(aq)}, and {ATP(aq) + pyruvate(aq) + phosphate(aq) = AMP(aq) + PEP(aq) + pyrophosphate(aq)} were also analyzed by using the chemical equilibrium model. These calculations yielded values of the equilibrium constants K and standard molar Gibbs free energy changes ΔrGmo for ionic reference reactions that correspond to the overall biochemical reactions. Combination of the standard molar reaction property values (K, ΔrH mo, and ΔrGmo) with the standard molar formation properties of the AMP, ADP, ATP, pyrophosphate, and pyruvate species led to values of the standard molar enthalpy ΔfHmo, and Gibbs free energy of formation ΔfGmo and the standard partial molar entropy Smo of the cAMP and PEP species. The thermochemical network appears to be reasonably well reinforced and thus lends some confidence to the accuracy of the calculated property values of the variety of species involved in the several reactions considered herein. Published by Elsevier Ltd.
- Goldberg, Robert N.,Tewari, Yadu B.
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p. 1809 - 1830
(2007/10/03)
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- Derivatives of L-adenosine and L-guanosine as substrates for human deoxycytidine kinase
-
A series of analogues of L-adenosine and of L-guanosine, including β- L-dA, β-L-Ado, β-L-araA, and β-L-dG, have been shown to be substrates of human deoxycytidine kinase thus demonstrating the complete lack of enantioselectivity of this enzyme.
- Gaubert,Gosselin,Imbach,Eriksson,Maury
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p. 857 - 860
(2007/10/03)
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- Interactions between aminocalixarenes and nucleotides or nucleic acids
-
Four calixarenes with (trimethylammonium)methyl groups at the phenyl rings in the upper rim were prepared. Association constants K with mononucleotides were determined in D2O by NMR shift titration, partially also by fluorescence competition titration using ANS as dye. The complexation free energies ΔG obtained with the derivatives of the calix[4]cone (AC4c) and the calix[4]-1,3-alternate (AC4a) conformation were similar, but increased from AMP (18 ± 1 kJ mol-1) to ADP (20 ± 1 kJ mol-1), to ATP (22 ± 1 kJ mol-1]. With the calix[6] derivative (AC6) the corresponding values were 22, 24, 27 kJ mol-1, with the calix[8] host (AC8) 24, 26, 28 kJ mol-1, respectively. The large contribution of salt bridging to the complexation was obvious from the ΔG difference between adenosine and e.g. AMP (with the calix[4]cone derivative 5.6 and 17.7 kJ mol-1, respectively). Affinity differences between different nucleobases increased moderately with the size of the macrocyclic host, e.g. ΔΔG between AMP and TMP was 1 kJ mor-1 with calix[4]cone, 2 kJ mor-1 with calix[6], and 3 kJ mol-1 with calix[8] compounds. The results are in line with computer simulated complex structures in which the nucleobase or sugar parts are only partially inserted into the calix cavity. This agrees with the observed complexation induced NMR shifts (CIS), which are small but increase with the ring size of the host. Noticeably the CIS values are substantially larger for much weaker bound nucleosides. Affinities of the four aminocalixarenes with double-stranded calf thymus (CT) DNA, with polydA*polydT and with polydG*polydC were characterized by ΔTm of the double-strand denaturation temperature and by fluorimetric assays using ethidium bromide (C50 values). The calix[4]cone derivative AC4c shows, due to the four positive charges converging at one side, the strongest effects. They surpass spermine although this also bears four protonated ammonium groups, indicating additional binding contributions from the phenyl moieties. The larger, more flexible calix[6]- and calix[8]-derivatives AC6 and AC8 show only small affinity increases in spite of their 6 or 8 positive charges. Preliminary molecular modeling studies indicate that based on the distances between the ammonium centers only partial contact of all centers to the groove phosphates can materialize. The ligands AC4c, AC4a and AC6 exhibit a remarkable preference for DNA in comparison to RNA mimics.
- Shi, Youheng,Schneider, Hans-Joerg
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p. 1797 - 1803
(2007/10/03)
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- Phenanthroline-containing macrocycles as multifunctional receptors for nucleotide anions. a thermodynamic and NMR study
-
The synthesis of the phenanthroline-containing macrocycle 2,6,10,14-tetraaza[15](2.9)cyclo(1,10)phenanthrolinophane (L1) is reported. L1 contains a tetraamine chain connecting the 2,9-positions of a phenanthroline unit. Protonation of L1 has been studied by means of potentiometric and 1H and 13C NMR techniques, allowing the determination of the basicity constants and of the stepwise protonation sites. The protonation features of L1 are compared with those of macrocycle 2,5,8,11-tetraaza[12](2,9)cyclophenanthrolinophane (L2), in which the amine groups are linked by ethylenic chains. Considering the [H4L1]4- and the [H4L2]4+ species, the acidic protons are located on the aliphatic nitrogens, while phenanthroline is not involved in protonation. Binding of diphosphate, triphosphate, ATP and ADP has been studied by means of potentiometry and 1H and 31P NMR. Both L1 and L2 behave as multifunctional receptors for the nucleotide anions at neutral or slight acidic pHs, giving 1 : 1 complexes. Charge-charge and hydrogen bonding interactions take place between the polyphosphate chain of nucleotides and the polyammonium groups of L1 and L2, while the adenine moiety gives charge-dipole interactions with the ammonium groups and π-stacking with the phenanthroline unit of the receptors. The high upfield displacements in the 1H NMR spectra exhibited by the adenine protons upon complexation by L1 suggest a partial inclusion of nucleotides inside the macrocyclic cavity.
- Bazzicalupi, Carla,Beconcini, Alessia,Bencini, Andrea,Fusi, Vieri,Giorgi, Claudia,Masotti, Andrea,Valtancoli, Barbara
-
p. 1675 - 1682
(2007/10/03)
-
- Use of Escherichia coli Polyphosphate Kinase for Oligosaccharide Synthesis
-
The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.
- Noguchi, Toshitada,Shiba, Toshikazu
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p. 1594 - 1596
(2007/10/03)
-
- Time-resolved infrared spectroscopy of intermediates and products from photolysis of 1-(2-nitrophenyl)ethyl phosphates: Reaction of the 2- nitrosoacetophenone byproduct with thiols
-
Rapid scan Fourier transform infrared (FTIR) spectroscopy and time- resolved single wavelength infrared (IR) spectroscopy have been applied to study the mechanism of photochemical release of adenosine 5'-triphosphate (ATP) from its P3-[1-(2-nitrophenyl)ethyl] ester (caged ATP). Bands arising from phosphate and non-phosphate vibrations characteristic of the aci-nitro anion intermediate and from free ATP and hyproducts of the nitrophenylethyl group have been assigned using 13C, 15N, and 18O isotopomers of caged ATP. Monitoring several of these bands using time-resolved single frequency IR spectroscopy confirms that release of ATP occurs in a single exponential process synchronous with the decay of the aci-nitro anion intermediate. Spectral characteristics of the reaction products arising from the l-(2- nitrophenyl)ethyl group in the absence and presence of dithiothreitol (DTT) have been determined. The major final byproduct from photolysis conducted in the presence of DTT is 3-methylanthranil. The mechanism of formation of this compound from 2-nitrosoacetophenone has been investigated in detail by a combination of spectroscopic, kinetic, and chemical methods and reconciled with earlier data. The byproduct species likely to be present on the time scale of most biological experiments using caged compounds is 2- hydroxylaminoacetophenone as a mixture of ring-chain tautomers.
- Barth, Andreas,Corrie, John E. T.,Gradwell, Michael J.,Maeda, Yashusi,M?ntele, Werner,Meier, Tanja,Trentham, David R.
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p. 4149 - 4159
(2007/10/03)
-
- 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.
- Schlattner, Uwe,Wagner, Edgar,Greppin, Hubert,Bonzon, Marc
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p. 589 - 594
(2007/10/03)
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- Photochemical release of ATP from "caged ATP" studied by time-resolved infrared spectroscopy
-
Rapid scan Fourier transform infrared (FTIR) spectroscopy and time-resolved single wavelength infrared (IR) spectroscopy have been used to follow the photochemical release of adenosine 5′-triphosphate (ATP) from P3-(1-(2-nitrophenyl)ethyl) adenosine 5′-triphosphate (caged ATP). Vibrational difference spectra for the formation first of the aci-nitro anion intermediate and subsequently of ATP and the byproduct(s) were obtained by rapid scan FTIR spectroscopy in the millisecond-to-second time domain. Vibrational modes of the phosphate groups of ATP and caged ATP in the range 1250-900 cm-1 could be assigned on the basis of triple and single 18O labeling in caged ATP at the terminal phosphate group and at the bridging oxygen between the terminal phosphate and the 1-(2-nitrophenyl)ethyl group, respectively. The rapid formation and subsequent decay of the aci-nitro anion intermediate were monitored by single-wavelength time-resolved IR spectroscopy at 1251 cm-1 (predominantly a PO2- mode of caged ATP and the aci-nitro intermediate). The appearance of the free γ-phosphate group of ATP was monitored at 1119 cm-1 (POs2- mode of ATP). Decay of the aci-nitro anion intermediate and formation of ATP were well fitted by single exponentials to give a mean rate constant of 218 ± 33 s-1 at pH 7 and 22 °C.
- Barth, Andreas,Hauser, Karin,M?ntele, Werner,Corrie, John E. T.,Trentham, David R.
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p. 10311 - 10316
(2007/10/02)
-
- A convenient method for the synthesis of ATP and Ap4A
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A bifunctional phosphorylating reagent, O-8-(5-chloroquinolyl) S-phenyl phosphorothioate (1) was employed for the synthesis of adenosine 5'- triphosphate(ATP) and diadenosine 5'-tetraphosphate(Ap4A) from adenosine 5'-phosphate(AMP) on a large scale.
- Fukuoka,Suda,Ishikawa,Hata
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p. 693 - 694
(2007/10/02)
-
- Mg(II) ION-MEDIATED CONVERSION OF MONO- AND OLIGONUCLEOTIDES TO 5'-POLYPHOSPHATES IN AQUEOUS SOLUTION
-
5'-Polyphosphates of mono- and oligonucleotides were prepared from the corresponding 5'-monophosphates with phosphorotriimidazolide or phosphorotribenzimidazolide mediated by Mg(II) or Mn(II) ion in aqueous solution.
- Sawai, Hiroaki,Inaba, Yoshiko,Hirano, Atsushi,Wakai, Hiromichi,Shimazu, Masamitsu
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p. 4801 - 4804
(2007/10/02)
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- Photolabile 1-(2-Nitrophenyl)ethyl Phosphate Esters of Adenine Nucleotide Analogues. Synthesis and Mechanism of Photolysis
-
A general method is described for preparing photolabile 1-(2-nitrophenyl)ethyl esters of phosphate and thiophosphate compounds.The method is based on selective alkylation of weakly ionizing phosphate groups by a new alkylating agent, 1-(2-nitrophenyl)diazoethane.ATP and the widely used structural analogues of ATP, 5'-adenylyl imidodiphosphate (ATP(β,γNH)) and adenosine 5'-(3-thiophsophate) (ATP(γS)), were alkylated on the terminal (γ) phosphate group.ATP(γS) was alkylated on oxygen or on sulfur in approximately equal amounts.Photolysis of P3-1-(2-nitrophenyl)ethyladenosine 5'-triphosphate, commonly called "caged ATP", was analyzed spectroscopically at pH values close to neutral in aqueous solvents by use of laser pulse photolysis.The kinetics of formation of the three products, ATP, 2-nitrosoacetophenone, and H+, were each monitored, as well as the kinetics of formation and decay of an intermediate presumed to be an aci-nitro compound (apparent ε406nm = 9.1 x 103 M-1 cm-1).For caged ATP in the presence of 3 mM MgCl2, and aci-nitro intermediate and H+ formed first at > 105 s-1 followed by the decay of the intermediate at 86 s-1 at pH 7.1, 21 deg C, and ionic strength 0.18 M.ATP, monitored by a biochemical assay, and 2-nitrosoacetophenone, monitored by a characteristic absorption band at 740 nm, were formed simultaneously with the decay of the intermediate under all conditions tested.The rate of decay of the aci-nitro intermediate was therefore used as a measure of the rate of release of the nucleotide analogues from their photolabile precursors.At pH 7.1, 0.18 M ionic strength, and 21 deg C the rate constants ranged from 35 to 250 s-1 and displayed a similar dependence on pH as caged ATP.The steady-state quantum yields of the 1-(2-nitrophenyl)ethyl phosphate esters were in the range 0.49 - 0.63.The deleterious effect of 2-nitrosoacetophenone on biological materials can be avoided by having thiols present.The reaction kinetics of dithiothreitol and 2-nitrosoacetophenone was described by a two-step process, the first step having a rate constant of 3.5 x 103 M-1 s-1 and the second 45 s-1 at pH 7.0, 21 deg C, and ionic strength 0.18 M.
- Walker, Jeffery W.,Reid, Gordon P.,McCray, James A.,Trentham, David R.
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p. 7170 - 7177
(2007/10/02)
-
- Synthesis of Nucleotide 5'-Diphosphates from 5'-O-Tosyl Nucleosides
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Procedures are described for the synthesis of nucleoside 5'-diphosphates, methanediphosphonates, and difluoromethanediphosphonates.The general strategy involves protection of the nucleosides as amidine, 2',3'-methoxymethylidene, and 3'-(tert-butyldimethylsilyl) derivatives prior to tosylation with tosyl chloride and (N,N-dimethylamino)pyridine.Deprotection, followed by displacement of the tosyl moiety with the tris(tetra-n-butylammonium) pyrophosphate, methanediphosphonate, or difluoromethanediphosphonate salts gave the desired products.The ammonium salts of the nucleotides were purified by flash chromatography on cellulose or medium pressure ion-exchange chromatography on DEAE Fractogel.Syntheses are reported for UDP (18), CDP (19), TDP (20), GDP (21), ADP (23), 2',3'-isopropylidene-ADP (22), adenosine 5'-methanediphosphonate (24), adenosine 5'-difluoromethanediphosphonate (25), and deoxyadenosine 5'-methanediphosphonate (27).In addition ATP (26) was prepared by treatment of 5'-O-tosyladenosine with tetrakis(tetra-n-butylammonium) thiophosphate.Yields for the displacement reactions ranged from 43percent to 93percent.
- Davisson, V. Jo,Davis, Darrell R.,Dixit, Vyas M.,Poulter, C. Dale
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p. 1794 - 1801
(2007/10/02)
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- OLIGONUCLEOTIDES AND NUCLEOTIDYLPEPTIDES. XXXII. SYNTHESIS AND HYDROLYTIC STABILITY OF AMINO ACID DERIVATIVES OF ADENOSINE 5'-DI(TRI)PHOSPHATES
-
The synthesis has been effected of the ethyl esters of adenosine -5'-diphospho(Pβ --> N)- and adenosine-5'-triphospho(Pγ --> N)-alanine.It has been shown that under the conditions of synthesis the serine analogues of ATP and ADP decompose.An investigation of the hydrolytic stability of the compounds synthesized has shown that they are unstable in acid and alkaline media.In an acid medium the phosphoramide bond is cleaved more rapidly than the phosphoric anhydride bond (in the case of ADP analogue), while in an alkaline medium the ester bond is saponified and the p hosphoric anhydride bond is cleaved.The ATP analogue is more labile both in acid and in alkaline media.
- Yuodka, B. A.,Sasnauskene, S. I.
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p. 479 - 483
(2007/10/02)
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- Direct Quantitative Analysis of Enzyme-Catalyzed Reactions by Two-Dimensional Nuclear Magnetic Resonance Spectroscopy: Adenylate Kinase and Phosphoglyceromutase
-
The back-transformation method for the analysis of two-dimensional nuclear magnetic resonance cross-relaxation and exchange data has been employed to obtain the rate constants of the phosphoglyceromutase and adenylate kinase catalyzed reactions in vitro.The results are in excellent agreement with those obtained by other methods.It is shown that a single two-dimensional experiment for each catalyzed reaction is sufficient to calculate the rate constants.The study of the adenylate kinase reaction demonstrates the potential of the method for direct analysis of higher order reactions without having to simplify them in terms of pseudo-first-order steps.
- Mendz, George L.,Robinson, Gae,Kuchel, Philip W.
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p. 169 - 173
(2007/10/02)
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- REVERSIBLE TRANSPHOSPHORYLATION AMONG ATP, ADP, AND AMP IN THE PRESENCE OF METHYLATED CYCLODEXTRIN
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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.
- Hattori, Kenjiro,Takahashi, Keiko
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p. 985 - 988
(2007/10/02)
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- KINETICS OF PROTON-TRANSPORT-COUPLED ATP SYNTHESIS IN CHLOROPLASTS. ACTIVATION OF THE ATPase BY AN ARTIFICALLY GENERATED DELTA pH AND DELTA PSI .
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The kinetics of proton transport coupled ATP synthesis at the chloroplast membrane was investigated upon energization of the membrane by an artificially generated DELTA pH and an electric potential difference, DELTA PSI . Using a rapid mixing system, rates of ATP synthesis were studied at short reaction times ( less than 150 ms) where all relevant parameters ( DELTA pH, DELTA PSI , substrate and product concentrations) remain practically constant at their initial values. Under these conditions it was found: 1. The maximal rate of ATP synthesis obtained under these artificial conditions is the same as that obtained by light-induced ATP synthesis. 2. The turnover number of the ATPase is 410 s** minus **1. 3. The rate of ATP synthesis depends in a sigmoidal way on the transmembrane electrochemical potential difference of protons, DELTA OVER BAR //H// plus , regardless of the relative contributions by DELTA pH and DELTA PSI .
- Graeber,Junesch,Schatz
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p. 599 - 608
(2007/10/02)
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- CONVENIENT SYNTHESES OF ADENOSINE 5'-DIPHOSPHATE, ADENOSINE 5'-METHYLENEDIPHOSPHONATE, AND ADENOSINE 5'-TRIPHOSPHATE
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Adenosine 5'-tosylate is converted to adenosine 5'-diphosphate (ADP), adenosine 5'-methylenediphosphonate, and adenosine 5'-triphosphate (ATP) in good yields by direct displacement with the appropriate inorganic salt.
- Dixit, Vyas M.,Poulter, C. Dale
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p. 4055 - 4058
(2007/10/02)
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