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Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum : Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum : Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

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TITTMANN, Kai, Georg WILLE, Ralph GOLBIK, Annett WEIDNER, Sandro GHISLA, Gerhard HÜBNER, 2005. Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum : Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair. In: Biochemistry. 44(40), pp. 13291-13303. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi051058z

@article{Tittmann2005Radic-7635, title={Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum : Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair}, year={2005}, doi={10.1021/bi051058z}, number={40}, volume={44}, issn={0006-2960}, journal={Biochemistry}, pages={13291--13303}, author={Tittmann, Kai and Wille, Georg and Golbik, Ralph and Weidner, Annett and Ghisla, Sandro and Hübner, Gerhard} }

<rdf:RDF xmlns:dcterms="http://purl.org/dc/terms/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#" xmlns:foaf="http://xmlns.com/foaf/0.1/" xmlns:void="http://rdfs.org/ns/void#" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/7635"> <dcterms:issued>2005</dcterms:issued> <dc:contributor>Golbik, Ralph</dc:contributor> <dcterms:bibliographicCitation>First publ. in: Biochemistry ; 44 (2005), 40. - S. 13291-13303</dcterms:bibliographicCitation> <foaf:homepage rdf:resource="http://localhost:8080/jspui"/> <dc:creator>Golbik, Ralph</dc:creator> <dcterms:title>Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum : Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair</dcterms:title> <dc:language>eng</dc:language> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dcterms:rights rdf:resource="https://creativecommons.org/licenses/by-nc-nd/2.0/legalcode"/> <dc:contributor>Weidner, Annett</dc:contributor> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:35:57Z</dc:date> <dc:contributor>Tittmann, Kai</dc:contributor> <dc:creator>Weidner, Annett</dc:creator> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/7635"/> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7635/1/Radical_Phosphate_Transfer_Mechanism_for_the_Thiamin_Diphosphate_and_FAD_Dependent_Pyruvate_Oxidase_from_Lactobacillus_plantarum.pdf"/> <dc:contributor>Hübner, Gerhard</dc:contributor> <dc:creator>Tittmann, Kai</dc:creator> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7635/1/Radical_Phosphate_Transfer_Mechanism_for_the_Thiamin_Diphosphate_and_FAD_Dependent_Pyruvate_Oxidase_from_Lactobacillus_plantarum.pdf"/> <dc:rights>deposit-license</dc:rights> <dc:creator>Hübner, Gerhard</dc:creator> <dcterms:abstract xml:lang="eng">The thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of -hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 Å, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s-1 and is kinetically gated by the preceding formation of the tetrahedral substrate-ThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s-1 and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FADox/FADsq (E1 = -37 mV) and FADsq/FADred (E2 = -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3'-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 102-103-fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosporolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.</dcterms:abstract> <dc:contributor>Ghisla, Sandro</dc:contributor> <dc:creator>Ghisla, Sandro</dc:creator> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <dc:contributor>Wille, Georg</dc:contributor> <dc:creator>Wille, Georg</dc:creator> <dc:format>application/pdf</dc:format> </rdf:Description> </rdf:RDF>

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