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Francisco García-carmona - One of the best experts on this subject based on the ideXlab platform.
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Kinetic characterization of cresolase activity of Streptomyces antibioticus tyrosinase
Enzyme and Microbial Technology, 2006Co-Authors: Esteban Orenes-piñero, Francisco García-carmona, Álvaro Sánchez-ferrer, Francisco García-carmona, Esteban Orenes-piñero, Álvaro Sánchez-ferrerAbstract:Abstract Streptomyces antibioticus tyrosinase was purified using PEG-8000/phosphate phase partitioning and ammonium sulfate fractionation between 0 and 60%, rendering a clear and stable enzyme compared with the black one that is usually obtained when only ammonium sulphate fractionation is used, and with a 93% of recovery. This enzyme shows both monophenolase (also termed cresolase) and diphenolase (catecholase) activities, confirming that it is a real tyrosinase. The monophenolase activity was characterized by a lag period, whose duration depends on the substrate concentration, the pH, and the presence of catalytic amounts of o -diphenol. The enzyme showed substrate inhibition for p -cresol, with a K M of 0.97 mM and a K si of 23 mM. By increasing the concentration of o -diphenols, it was possible to evaluate the enzyme Activation Constant, K act , which showed a value of 0.2 μM. The diphenolase activity was kinetically characterized with 4-methylcathechol, showed an optimal pH at 6.5 and a K M of 1.3 mM.
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Kinetic properties of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps: effect of inhibitors and activators.
Journal of agricultural and food chemistry, 2005Co-Authors: Manuela Pérez-gilabert, † Isabel Sánchez-felipe, And Asunción Morte, Francisco García-carmonaAbstract:There is very little information available on the kinetic characteristics of fungal lipoxygenases (LOXs) because most data on the mechanism of this enzyme concern soybean LOX. In this paper, the kinetic properties of LOX from Terfezia claveryi Chatin ascocarps were studied for the first time. The enzyme did not show the “substrate aggregation-dependent activity” described for other LOXs and presented a Km for linoleic acid of 41 μM at pH 7.0. The effect of different inhibitors was also studied. The enzyme presented the characteristic lag phase of other LOXs, and the influence of different factors on its duration was analyzed. The lag period was reduced not only by the product of the reaction (13-HPOD) but also by 9-HPOD. Calculation of the Activation Constant is proposed for the first time as a useful tool for the characterization of LOX because this method makes it possible to quantify the effectiveness of different hydroperoxides as LOX activators. The Activation Constants obtained were 0.3 and 6.4 μM f...
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A kinetic study of p-cresol oxidation by quince fruit polyphenol oxidase.
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Esteban Orenes-piñero, Francisco García-carmona, Álvaro Sánchez-ferrerAbstract:The monophenolase activity of quince pulp polyphenol oxidase was characterized by extracting samples using a combination of a two-phase partition step in Triton X-114, followed by a PEG 8000/phosphate partition step, and a final ammonium sulfate fractionation between 30 and 75%. The purification method avoids the loss of cresolase activity described in another quince pulp polyphenol oxidase. The activity was characterized by a lag period, whose duration depended on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme Activation Constant, Kact, which showed a value of 4.5 μM for 4-methylcatechol. A general kinetic mechanism for this enzyme is used to explain the loss of activity that normally occurs during quince pulp polyphenol oxidase purification. Keywords: Quince; polyphenol oxidase; monophenolase; lag period
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Monophenolase activity of latent Terfezia claveryi tyrosinase: Characterization and histochemical localization.
Physiologia plantarum, 2001Co-Authors: Manuela Pérez-gilabert, Asunción Morte, Mario Honrubia, Francisco García-carmonaAbstract:The monophenolase activity of Terfezia claveryi tyrosinase (EC 1.14.18.1) is described for the first time. This enzyme is fully latent and can only be detected if SDS is present in the reaction medium. Monophenolase activity was localized within the ascocarp using histochemical techniques. A detailed kinetic study of the parameters affecting this activity has been carried out. Both the characteristic lag period and the steady-state rate are affected by pH and the enzyme and substrate concentrations. The presence of catalytic concentrations of o-diphenols affected the lag period but not the steady-state rate. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme Activation Constant, K act , which showed a value of 7.2 μM. The experimental results are compatible with the mechanism previously described for tyrosinases from other sources.
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Cresolase activity of potato tuber partially purified in a two-phase partition system
Journal of Agricultural and Food Chemistry, 1993Co-Authors: Álvaro Sánchez-ferrer, Francisco. Laveda, Francisco García-carmonaAbstract:The cresolase activity of partially purified potato polyphenol oxidase extracted by a two-phase partition method in Triton X-114 has been characterized without using ascorbic acid. The purification method avoids the loss of cresolase activity described by other potato polyphenol oxidases. The activity was characterized by a lag period, whose duration depended on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme Activation Constant, K act , which showed a value of 4.5 μM
Manuela Pérez-gilabert - One of the best experts on this subject based on the ideXlab platform.
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Kinetic properties of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps: effect of inhibitors and activators.
Journal of agricultural and food chemistry, 2005Co-Authors: Manuela Pérez-gilabert, † Isabel Sánchez-felipe, And Asunción Morte, Francisco García-carmonaAbstract:There is very little information available on the kinetic characteristics of fungal lipoxygenases (LOXs) because most data on the mechanism of this enzyme concern soybean LOX. In this paper, the kinetic properties of LOX from Terfezia claveryi Chatin ascocarps were studied for the first time. The enzyme did not show the “substrate aggregation-dependent activity” described for other LOXs and presented a Km for linoleic acid of 41 μM at pH 7.0. The effect of different inhibitors was also studied. The enzyme presented the characteristic lag phase of other LOXs, and the influence of different factors on its duration was analyzed. The lag period was reduced not only by the product of the reaction (13-HPOD) but also by 9-HPOD. Calculation of the Activation Constant is proposed for the first time as a useful tool for the characterization of LOX because this method makes it possible to quantify the effectiveness of different hydroperoxides as LOX activators. The Activation Constants obtained were 0.3 and 6.4 μM f...
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Monophenolase activity of latent Terfezia claveryi tyrosinase: Characterization and histochemical localization.
Physiologia plantarum, 2001Co-Authors: Manuela Pérez-gilabert, Asunción Morte, Mario Honrubia, Francisco García-carmonaAbstract:The monophenolase activity of Terfezia claveryi tyrosinase (EC 1.14.18.1) is described for the first time. This enzyme is fully latent and can only be detected if SDS is present in the reaction medium. Monophenolase activity was localized within the ascocarp using histochemical techniques. A detailed kinetic study of the parameters affecting this activity has been carried out. Both the characteristic lag period and the steady-state rate are affected by pH and the enzyme and substrate concentrations. The presence of catalytic concentrations of o-diphenols affected the lag period but not the steady-state rate. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme Activation Constant, K act , which showed a value of 7.2 μM. The experimental results are compatible with the mechanism previously described for tyrosinases from other sources.
Claudiu T Supuran - One of the best experts on this subject based on the ideXlab platform.
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Activation studies of the γ-carbonic anhydrases from the antarctic marine bacteria pseudoalteromonas haloplanktis and colwellia psychrerythraea with amino acids and amines
Marine drugs, 2019Co-Authors: Andrea Angeli, Sonia Del Prete, Clemente Capasso, Sameh M. Osman, Zeid A. Alothman, William A. Donald, Claudiu T SupuranAbstract:The γ-carbonic anhydrases (CAs, EC 4.2.1.1) present in the Antarctic marine bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea, herein referred to as PhaCA and CpsCA, respectively, were investigated for their Activation with a panel of 24 amino acids and amines. Both bacteria are considered Antarctic models for the investigation of photosynthetic and metabolic pathways in organisms adapted to live in cold seawater. PhaCA was much more sensitive to Activation by these compounds compared to the genetically related enzyme CpsCA. The most effective PhaCA activators were d-Phe, l-/d-DOPA, l-Tyr and 2-pyridyl-methylamine, with the Activation Constant KA values of 0.72–3.27 µM. d-His, l-Trp, d-Tyr, histamine, dopamine, serotonin anddicarboxylic amino acids were also effective activators of PhaCA, with KA values of 6.48–9.85 µM. CpsCA was activated by d-Phe, d-DOPA, l-Trp, l-/d-Tyr, 4-amino-l-Phe, histamine, 2-pyridyl-methylamine and l-/d-Glu with KA values of 11.2–24.4 µM. The most effective CpsCA activator was l-DOPA (KA of 4.79 µM). Given that modulators of CAs from Antarctic bacteria have not been identified and investigated in detail for their metabolic roles to date, this research sheds some light on these poorly understood processes.
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Carbonic anhydrase activators. The first Activation study of a coral secretory isoform with amino acids and amines.
Bioorganic & medicinal chemistry, 2010Co-Authors: Anthony Bertucci, Daniela Vullo, Didier Zoccola, Sylvie Tambutté, Claudiu T SupuranAbstract:The activity of the coral Stylophora pystillata secretory carbonic anhydrase STPCA has been tested in presence of amino acids and amines. All the investigated compounds showed a positive, activating effect on k(cat) and have been separated in weak (K(A) in the range of 21-126 microM), medium (10.1-19 microM) and strong enzyme activators (K(A) of 0.18-3.21 microM). D-DOPA was found to be the best coral enzyme activator, with an Activation Constant K(A) of 0.18 microM. This enhancement of STPCA activity, as well as previous enzyme inhibition results, might now be tested on living organisms to better understand the role played by these enzymes in the coral calcification processes.
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Carbonic anhydrase activators: Activation of human isozymes I, II and IX with phenylsulfonylhydrazido l-histidine derivatives.
Bioorganic & Medicinal Chemistry Letters, 2009Co-Authors: Marie-rose Abdo, Andrea Scozzafava, Daniela Vullo, Mohamed-chiheb Saada, Jean-louis Montero, Jean-yves Winum, Claudiu T SupuranAbstract:Abstract Activation of the human carbonic anhydrase (CA, EC 4.2.1.1) isozymes I, II (cytosolic) and IX (transmembrane, tumor-associated isoform) with a series of arylsulfonylhydrazido- l -histidines incorporating 4-substituted-phenyl, pentafluorophenyl- and β-naphthyl moieties was investigated. The compounds showed a weak hCA I Activation profile, but were more efficient as hCA II and IX activators. The 4-iodophenyl-substituted derivative behaved as a strong and isozyme selective hCA II activator, with an Activation Constant of 0.21 μM. This is the first isoform-selective, potent CA activator reported to date.
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Carbonic anhydrase activators : Activation of the β-carbonic anhydrase Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids
Bioorganic & medicinal chemistry letters, 2009Co-Authors: Semra Isik, Andrea Scozzafava, Alessio Innocenti, Feray Kockar, Meltem Aydin, Oktay Arslan, Ozen Ozensoy Guler, Claudiu T SupuranAbstract:Abstract The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its Activation with amines and amino acids. scCA was poorly activated by amino acids such as l -/ d -His, Phe, DOPA, Trp (KAs of 82–90 μM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (KAs of 10.2–21.3 μM). The best activator was l -adrenaline, with an Activation Constant of 0.95 μM. This study may help to better understand the catalytic/Activation mechanisms of the β-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.
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carbonic anhydrase activators l adrenaline plugs the active site entrance of isozyme ii activating better isoforms i iv va vii and xiv
Bioorganic & Medicinal Chemistry Letters, 2007Co-Authors: Claudia Temperini, Andrea Scozzafava, Alessio Innocenti, Antonio Mastrolorenzo, Claudiu T SupuranAbstract:Abstract The Activation of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with l -adrenaline and histamine has been investigated by kinetic and X-ray crystallographic studies. l -Adrenaline behaves as a potent activator of isozyme CA I (Activation Constant of 90 nM), being a much weaker activator of isozyme CA II (Activation Constant of 96 μM). Isoforms CA IV, VA, VII, and XIV were activated by l -adrenaline with KAs in the range of 36–63 μM. The X-ray crystal structure of the CA II– l -adrenaline adduct revealed that the activator plugs the entrance of the active site cavity, obstructing it almost completely.
Vern L Schramm - One of the best experts on this subject based on the ideXlab platform.
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transition state analysis of a vmax mutant of amp nucleosidase by the application of heavy atom kinetic isotope effects
Biochemistry, 1991Co-Authors: David W Parkin, Frank Mentch, Grace A Banks, Benjamin A Horenstein, Vern L SchrammAbstract:The transition state of the V{sub max} mutant of AMP nucleosidase from Azotobacter vinelandii has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the K{sub m} for substrate, the Activation Constant for MgATP, and the K{sub i} for formycin 5{prime}-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s{sup {minus}1}. The kinetic isotope effects were measured with the substrates (1{prime}-{sup 3}H)AMP, (2{prime}-{sup 2}H)AMP, (9-{sup 15}N)AMP, and (1{prime},9-{sup 14}C, {sup 15}N)AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. Transition-state analysis using bond-energy and bond-order vibrational analysis indicated that the transition state for the mutant enzyme has a similar position in the reaction coordinate compared to that for the normal enzyme. The mutant enzyme is less effective in stabilizing the carbocation-like intermediate and in the ability to protonate N7 ofmore » adenine to create a better leaving group. This altered transition-state structure was confirmed by an altered substrate specificity for the mutant protein.« less
Álvaro Sánchez-ferrer - One of the best experts on this subject based on the ideXlab platform.
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Kinetic characterization of cresolase activity of Streptomyces antibioticus tyrosinase
Enzyme and Microbial Technology, 2006Co-Authors: Esteban Orenes-piñero, Francisco García-carmona, Álvaro Sánchez-ferrer, Francisco García-carmona, Esteban Orenes-piñero, Álvaro Sánchez-ferrerAbstract:Abstract Streptomyces antibioticus tyrosinase was purified using PEG-8000/phosphate phase partitioning and ammonium sulfate fractionation between 0 and 60%, rendering a clear and stable enzyme compared with the black one that is usually obtained when only ammonium sulphate fractionation is used, and with a 93% of recovery. This enzyme shows both monophenolase (also termed cresolase) and diphenolase (catecholase) activities, confirming that it is a real tyrosinase. The monophenolase activity was characterized by a lag period, whose duration depends on the substrate concentration, the pH, and the presence of catalytic amounts of o -diphenol. The enzyme showed substrate inhibition for p -cresol, with a K M of 0.97 mM and a K si of 23 mM. By increasing the concentration of o -diphenols, it was possible to evaluate the enzyme Activation Constant, K act , which showed a value of 0.2 μM. The diphenolase activity was kinetically characterized with 4-methylcathechol, showed an optimal pH at 6.5 and a K M of 1.3 mM.
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A kinetic study of p-cresol oxidation by quince fruit polyphenol oxidase.
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Esteban Orenes-piñero, Francisco García-carmona, Álvaro Sánchez-ferrerAbstract:The monophenolase activity of quince pulp polyphenol oxidase was characterized by extracting samples using a combination of a two-phase partition step in Triton X-114, followed by a PEG 8000/phosphate partition step, and a final ammonium sulfate fractionation between 30 and 75%. The purification method avoids the loss of cresolase activity described in another quince pulp polyphenol oxidase. The activity was characterized by a lag period, whose duration depended on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme Activation Constant, Kact, which showed a value of 4.5 μM for 4-methylcatechol. A general kinetic mechanism for this enzyme is used to explain the loss of activity that normally occurs during quince pulp polyphenol oxidase purification. Keywords: Quince; polyphenol oxidase; monophenolase; lag period
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Cresolase activity of potato tuber partially purified in a two-phase partition system
Journal of Agricultural and Food Chemistry, 1993Co-Authors: Álvaro Sánchez-ferrer, Francisco. Laveda, Francisco García-carmonaAbstract:The cresolase activity of partially purified potato polyphenol oxidase extracted by a two-phase partition method in Triton X-114 has been characterized without using ascorbic acid. The purification method avoids the loss of cresolase activity described by other potato polyphenol oxidases. The activity was characterized by a lag period, whose duration depended on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme Activation Constant, K act , which showed a value of 4.5 μM
