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John E. Cronan - One of the best experts on this subject based on the ideXlab platform.
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Conversion of Escherichia coli Pyruvate Oxidase to an ‘α-ketobutyrate Oxidase’
Biochemical Journal, 2000Co-Authors: Ying-ying Chang, John E. CronanAbstract:Escherichia coli Pyruvate Oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both Pyruvate and 2-oxobutanoate ('alpha-ketobutyrate'), although Pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, Pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380 had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable Pyruvate Oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in K(m) for Pyruvate and a 10-fold lower V(max) with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of Pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.
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Sulfhydryl chemistry detects three conformations of the lipid binding region of Escherichia coli Pyruvate Oxidase.
Biochemistry, 1997Co-Authors: Ying-ying Chang, John E. CronanAbstract:Site-specific disulfide cross-linking experiments detected a conformational change within the C-terminal segment of Escherichia coli Pyruvate Oxidase (PoxB), a lipid-activated homotetrameric enzyme, upon substrate binding [Chang, Y.-Y., & Cronan, J. E., Jr. (1995) J. Biol. Chem. 270, 7896−7901]. The C-terminal lipid binding regions were cross-linked only in the presence of the substrate, Pyruvate, and the thiamine pyrophosphate cofactor, indicating close proximity of a pair of C termini. We have now systematically substituted cysteine at 18 additional amino acid positions within the C-terminal region to obtain a panel of 21 proteins each having a single residue changed to cysteine. These proteins have been studied by disulfide cross-linking and by accessibility of the cysteine side chain to a variety of sulfhydryl agents. In the absence of Pyruvate, the cysteine residues of the modified PoxB proteins failed to form disulfide bonds, generally failed to react with a large and rigid hydrophilic sulfhydryl re...
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Detection by Site-specific Disulfide Cross-linking of a Conformational Change in Binding of Escherichia coli Pyruvate Oxidase to Lipid Bilayers
The Journal of biological chemistry, 1995Co-Authors: Ying-ying Chang, John E. CronanAbstract:Abstract Escherichia coli Pyruvate Oxidase, a peripheral membrane homotetrameric flavoprotein, exposes its C-terminal lipid binding site in the presence of substrate Pyruvate and co-factor thiamine pyrophosphate Mg and binds tightly to phospholipid bilayers during catalysis. Using site-specific disulfide cross-linking, we demonstrate that disulfide cross-links are formed between C termini of D560C Pyruvate Oxidase and that the degree of cross-linking is greatly increased by the presence of substrate and co-factors indicating a conformational change that results in juxtaposition of two subunit C termini. The cross-linked Oxidase is enzymatically active and remains able to associate with lipid micelles. These results argue strongly that lipid bilayer binding of Pyruvate Oxidase involves pairing of the C termini of two subunits.
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Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase. An enzyme of the acetohydroxy acid synthase-Pyruvate Oxidase family.
The Journal of biological chemistry, 1993Co-Authors: Ying-ying Chang, Ai Yu Wang, John E. CronanAbstract:Abstract Glyoxylate carboligase (Gcl) (EC 4.1.1.47) of Escherichia coli catalyzes the condensation of two molecules of glyoxylate to give tartronic semialdehyde, a key intermediate in glyoxylate catabolism. We report the cloning, genomic location, and DNA sequence of the gene (called gcl) encoding E. coli Gcl and isolation of mutants lacking the enzyme. Gcl is a protein of 593 amino acid residues (64,738 Da) that has a high level (30%) of sequence similarity to the acetohydroxy acid synthases (AHAS) of branched chain amino acid synthetic pathway. Significant sequence identity (26%) was also observed with E. coli Pyruvate Oxidase, a redox flavoprotein, previously shown to be related to the AHAS enzymes (Chang, Y.-Y., and Cronan, J. E., Jr. (1988) J. Bacteriol. 170, 3937-3945). Consistent with a grouping of Gcl with the AHAS and Pyruvate Oxidase enzymes. Gcl contains a quinone binding site as well as binding site for thiamine pyrophosphate and FAD. We also found that a gene (orf258) immediately downstream of the gcl gene encoded a protein (Orf258) of 258 residues. Although the gene organization of gcl and orf258 is analogous to that of the ilv gene operons which encode the E. coli AHAS isozyme large and small subunits, Orf258 does not function as a Gcl subunit. Moreover, disruption of the chromosomal copy of orf258 did not affect growth on glyoxylate or glycolate.
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Role of the tetrameric structure of Escherichia coli Pyruvate Oxidase in enzyme activation and lipid binding.
The Journal of biological chemistry, 1991Co-Authors: Ai Yu Wang, Ying-ying Chang, John E. CronanAbstract:Pyruvate Oxidase of Escherichia coli, an enzyme greatly activated by phospholipids, is a tetramer of a Mr 62,000 subunit. We have utilized the differing electrophoretic mobilities of several mutant Oxidases on native polyacrylamide gels to study the role of the quaternary structure of the enzyme in the activation process. We found that when two poxB gene alleles coexisted in cells, heterotetrameric species were formed in addition to homotetramers. The concentration of each tetrameric species varied according to the concentration of the different subunits present, and the distribution seemed virtually identical to those expected from random mixing. We showed that the intrinsic activity of Pyruvate Oxidase was not affected by interactions among the four subunits. However, binding of the enzyme to lipids, a property required for function in vivo, required that a tetramer contain at least two subunits capable of lipid binding. Our data fit the model proposed previously (Grabau, C., Chang, Y.-Y., and Cronan, J. E., Jr. (1989) J. Biol. Chem. 264, 12510-12519) in which the carboxyl termini of two subunits interact to form a functional lipid-binding domain. We also have detected Oxidase activity in a form of Oxidase of unusually high electrophoretic mobility. This form seems to be either a monomeric or a dimeric form (more probably the former) of the Oxidase subunit.
Gunther Kern - One of the best experts on this subject based on the ideXlab platform.
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activation of thiamin diphosphate and fad in the phosphatedependent Pyruvate Oxidase fromlactobacillus plantarum
Journal of Biological Chemistry, 1998Co-Authors: Kai Tittmann, Daniela Proske, Michael Spinka, Sandro Ghisla, Rainer Rudolph, Gerhard Hubner, Gunther KernAbstract:The phosphate- and oxygen-dependent Pyruvate Oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of Pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in Pyruvate Oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-Pyruvate Oxidase ternary complex requires a second step, which was detected at low temperature.
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Activation of thiamin diphosphate and FAD in the phosphatedependent Pyruvate Oxidase from Lactobacillus plantarum
The Journal of biological chemistry, 1998Co-Authors: Kai Tittmann, Daniela Proske, Michael Spinka, Sandro Ghisla, Rainer Rudolph, Gerhard Hubner, Gunther KernAbstract:The phosphate- and oxygen-dependent Pyruvate Oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of Pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in Pyruvate Oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-Pyruvate Oxidase ternary complex requires a second step, which was detected at low temperature.
Kai Tittmann - One of the best experts on this subject based on the ideXlab platform.
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Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated Pyruvate Oxidase from Escherichia coli
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2008Co-Authors: Annett Weidner, Piotr Neumann, Georg Wille, Milton T. Stubbs, Kai TittmannAbstract:The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme Pyruvate Oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Δ23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Δ23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 A. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P43212 with two monomers per asymmetric unit, the crystals of Δ23 EcPOX belong to the orthorhombic space group P212121 and contain 12 monomers per asymmetric unit.
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Phosphate mediates electron transfer in Pyruvate Oxidase from Lactobacillus plantarum
2002Co-Authors: Kai Tittmann, Gerhard Hubner, Ralph Golbik, Sandro GhislaAbstract:Introduction Pyruvate Oxidase from Lactobacillus plantarum (LpPOX, EC 1.2.3.3) is homotetrameric flavoenzyme with a subunit molecular mass of 65.5 kDa composed 603 residues. Each subunit contains one tightly and noncovalently bound FA thiamin diphosphate (ThDP), the biologically active form of vitamin BI. and Mgz+ anchoring the diphosphate moiety of ThDP. In the presence of phosphate and oxyg LpPOX catalyses the oxidativedecarboxylation of Pyruvate yielding carbon dioxi acetylphosphate and hydrogen peroxide. Tbe catalytic cycle of LpPOX comprises i) the deprotonation at the C2 of ThDP, the covalent binding of Pyruvate to ThDP, iii) the decarboxylation of the then formed 2-1actyl-ThDP (LThDP) intermediate to yield the carbanion/enamine .of hydroxyethyl-ThDP (HEThDP), iv) an intramolecular two step electron transfer fr HEThDP to FAD and finally v) the reoxidation of FADHz by oxygen and vi) phosphorolysislhydrolysis of the 2-acetyl-ThDP (AcThDP) intermediate (1). A kinetic analysis of single steps of catalysis using FAD absorbance revealed phosphorolysis of the AcThDP intermediate as weil as the reoxidation of the redu FAD to be partially rate-limiting. In the presence of phosphate no transient rad FAD species can be observed in the course of the reductive half-reaction (2). Here, we present stopped flow and titrimetric results of the influence of the subst phosphate on the reductive and oxidative half-reaction as weil as the stabilisatior radical FAD species in the enzyme.
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Mechanism of elementary catalytic steps of Pyruvate Oxidase from Lactobacillus plantarum.
Biochemistry, 2000Co-Authors: Kai Tittmann, Sandro Ghisla, Ralph Golbik, Gerhard HubnerAbstract:Single steps in the catalytic cycle of Pyruvate Oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of Pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of Pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.
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activation of thiamin diphosphate and fad in the phosphatedependent Pyruvate Oxidase fromlactobacillus plantarum
Journal of Biological Chemistry, 1998Co-Authors: Kai Tittmann, Daniela Proske, Michael Spinka, Sandro Ghisla, Rainer Rudolph, Gerhard Hubner, Gunther KernAbstract:The phosphate- and oxygen-dependent Pyruvate Oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of Pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in Pyruvate Oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-Pyruvate Oxidase ternary complex requires a second step, which was detected at low temperature.
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Activation of thiamin diphosphate and FAD in the phosphatedependent Pyruvate Oxidase from Lactobacillus plantarum
The Journal of biological chemistry, 1998Co-Authors: Kai Tittmann, Daniela Proske, Michael Spinka, Sandro Ghisla, Rainer Rudolph, Gerhard Hubner, Gunther KernAbstract:The phosphate- and oxygen-dependent Pyruvate Oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of Pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in Pyruvate Oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-Pyruvate Oxidase ternary complex requires a second step, which was detected at low temperature.
Isao Karube - One of the best experts on this subject based on the ideXlab platform.
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An automatic flow-injection analysis system for determining phosphate ion in river water using Pyruvate Oxidase G (from Aerococcus viridans).
Talanta, 1999Co-Authors: Hideaki Nakamura, Kazunori Ikebukuro, Yoshiko Arikawa, Hiroko Tanaka, Mami Hasegawa, Yuzo Masuda, Yoko Nomura, Isao KarubeAbstract:An automated flow-injection system combining a Pyruvate Oxidase reaction and chemiluminescence for the detection of phosphate ion in river water has been developed. In this research, we used Pyruvate Oxidase G (PyrOxG), from Aerococcus viridans, immobilizing it on N-hydroxysuccinicacidimido beads without a cross-linker. In this sensor system, which was constructed as a trial system of desktop type, the temperature was precisely controlled. After the sensor system was optimized, a calibration curve was obtained with a detection limit of 96 nM phosphate ion, a range between 96 nM and 32 microM phosphate ion, and a relative standard deviation of 2.3% (n=5) at 25 degrees C. The sensitivity of this sensor was sufficient to determine the maximal permissible phosphate-ion concentration in the environmental waters of Japan (0.32 microM). In addition, the sensor could determine the calibration curves between 0.16 and 32 microM phosphate ion (five points, n=3; averaged correlation, r=1.00) for at least 2 weeks, demonstrating enough stability for practical use. Furthermore, we investigated the influence on the sensor response of dissolved substances in river water such as metal ions, heavy metal ions, inorganic ions, and organic compounds. Treatment with activated carbon could improve the response of the sensor when inhibited by dissolved substances in river water, except for manganese ion and uric acid. The sensor system could determine the concentrations of phosphate ion in various samples of river water from the Tone River. The results obtained by this sensor system and the modified molybdenum blue method were compared, and good correlation (r=0.94) was obtained.
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A chemiluminescent FIA biosensor for phosphate ion monitoring using Pyruvate Oxidase.
Biosensors and Bioelectronics, 1997Co-Authors: Hideaki Nakamura, Masayasu Suzuki, Kenji Hayashi, Scott Mcniven, Kazunori Ikebukuro, Isao Karube, Hiroyuki Yamamoto, Izumi KuboAbstract:Abstract We have constructed an automatic phosphate ion sensing system for the quality control of drinking water. The analyte was detected using the phosphate ion-dependent Pyruvate Oxidase reaction and the hydrogen peroxide produced was detected by luminol chemiluminescence catalyzed by Arthromyces ramosus perOxidase. We obtained a detection limit of 0·16 μ M phosphate ion (5 ppb phosphorus) and it was possible to detect 0·32 μ M phosphate ion for 48 days using Pyruvate Oxidase immobilized on Chitopearl BCW-2601 beads. An excellent correlation ( r 2 =1·00) was obtained between the results obtained using our phosphate ion sensor and those using a modified Molybdenum Blue method.
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Phosphate sensing system using Pyruvate Oxidase and chemiluminescence detection
Biosensors and Bioelectronics, 1996Co-Authors: Kazunori Ikebukuro, Izumi Kubo, Masayasu Suzuki, Isao Karube, Hideaki Wakamura, Masako Inagawa, Takako Sugawara, Yoshiko Arikawa, Toshifumi TakeuchiAbstract:Abstract A flow injection phosphate analysis system based on an enzymic reaction and a subsequent luminol chemiluminescence reaction has been developed. The system consists of an immobilized Pyruvate Oxidase column, mixing chamber for the chemiluminescent reaction and a photomultiplier. The H 2 O 2 generated by the reaction of phosphate and Pyruvate Oxidase then reacts with luminol and horseradish perOxidase and the consequent chemiluminescence is detected using a photomultiplier. This system is capable of the rapid determination of phosphate, the time required for one measurement cycle being approximately 3 min. A linear response was observed from 4.8 to 160 μM phosphate.
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Phosphate Sensor Composed from Immobilized Pyruvate Oxidase and an Oxygen Electrode
Analytical Letters, 1991Co-Authors: Izumi Kubo, Masako Inagawa, Takako Sugawara, Yoshiko Arikawa, Isao KarubeAbstract:Abstract A phosphate sensor based on amperometric determination was constructed with immobilized Pyruvate Oxidase and an oxygen electrode. A rapid, simple and sensitive determination of phosphate was performed with the sensor. The determination time was 7min. The linearity range of this sensor was 12 – 80μM.
Gerhard Hubner - One of the best experts on this subject based on the ideXlab platform.
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Phosphate mediates electron transfer in Pyruvate Oxidase from Lactobacillus plantarum
2002Co-Authors: Kai Tittmann, Gerhard Hubner, Ralph Golbik, Sandro GhislaAbstract:Introduction Pyruvate Oxidase from Lactobacillus plantarum (LpPOX, EC 1.2.3.3) is homotetrameric flavoenzyme with a subunit molecular mass of 65.5 kDa composed 603 residues. Each subunit contains one tightly and noncovalently bound FA thiamin diphosphate (ThDP), the biologically active form of vitamin BI. and Mgz+ anchoring the diphosphate moiety of ThDP. In the presence of phosphate and oxyg LpPOX catalyses the oxidativedecarboxylation of Pyruvate yielding carbon dioxi acetylphosphate and hydrogen peroxide. Tbe catalytic cycle of LpPOX comprises i) the deprotonation at the C2 of ThDP, the covalent binding of Pyruvate to ThDP, iii) the decarboxylation of the then formed 2-1actyl-ThDP (LThDP) intermediate to yield the carbanion/enamine .of hydroxyethyl-ThDP (HEThDP), iv) an intramolecular two step electron transfer fr HEThDP to FAD and finally v) the reoxidation of FADHz by oxygen and vi) phosphorolysislhydrolysis of the 2-acetyl-ThDP (AcThDP) intermediate (1). A kinetic analysis of single steps of catalysis using FAD absorbance revealed phosphorolysis of the AcThDP intermediate as weil as the reoxidation of the redu FAD to be partially rate-limiting. In the presence of phosphate no transient rad FAD species can be observed in the course of the reductive half-reaction (2). Here, we present stopped flow and titrimetric results of the influence of the subst phosphate on the reductive and oxidative half-reaction as weil as the stabilisatior radical FAD species in the enzyme.
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Mechanism of elementary catalytic steps of Pyruvate Oxidase from Lactobacillus plantarum.
Biochemistry, 2000Co-Authors: Kai Tittmann, Sandro Ghisla, Ralph Golbik, Gerhard HubnerAbstract:Single steps in the catalytic cycle of Pyruvate Oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of Pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of Pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.
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activation of thiamin diphosphate and fad in the phosphatedependent Pyruvate Oxidase fromlactobacillus plantarum
Journal of Biological Chemistry, 1998Co-Authors: Kai Tittmann, Daniela Proske, Michael Spinka, Sandro Ghisla, Rainer Rudolph, Gerhard Hubner, Gunther KernAbstract:The phosphate- and oxygen-dependent Pyruvate Oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of Pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in Pyruvate Oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-Pyruvate Oxidase ternary complex requires a second step, which was detected at low temperature.
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Activation of thiamin diphosphate and FAD in the phosphatedependent Pyruvate Oxidase from Lactobacillus plantarum
The Journal of biological chemistry, 1998Co-Authors: Kai Tittmann, Daniela Proske, Michael Spinka, Sandro Ghisla, Rainer Rudolph, Gerhard Hubner, Gunther KernAbstract:The phosphate- and oxygen-dependent Pyruvate Oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of Pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in Pyruvate Oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-Pyruvate Oxidase ternary complex requires a second step, which was detected at low temperature.
