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
| Autor: | Ralph Golbik, Georg Wille, Kai Tittmann, Sandro Ghisla, Annett Weidner, Gerhard Hübner |
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| Rok vydání: | 2005 |
| Předmět: |
Magnetic Resonance Spectroscopy
Time Factors Free Radicals Stereochemistry Pyruvate Oxidase Radical Electrons Flavin group Photochemistry Biochemistry Catalysis Cofactor Phosphates chemistry.chemical_compound Electron transfer ddc:570 Flavins Pyruvic Acid Fluorescence Resonance Energy Transfer Pyruvate oxidase Phosphorolysis Flavin adenine dinucleotide Models Statistical biology Temperature Hydrogen-Ion Concentration Phosphate Oxygen Kinetics Models Chemical chemistry Spectrophotometry Flavin-Adenine Dinucleotide Solvents biology.protein Thermodynamics Thiamine Pyrophosphate Oxidation-Reduction Lactobacillus plantarum |
| Zdroj: | Biochemistry. 44:13291-13303 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi051058z |
| Popis: | 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 alpha-hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 A, 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 FAD(ox)/FAD(sq) (E1 = -37 mV) and FAD(sq)/FAD(red) (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 10(2)-10(3)-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 phosphorolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry. |
| Databáze: | OpenAIRE |
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