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Mechanism of Elementary Catalytic Steps of Pyruvate Oxidase from Lactobacillus plantarum

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2000

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Tittmann, Kai
Golbik, Ralph
Hübner, Gerhard

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Biochemistry. 2000, 39(35), pp. 10747-10754. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi0004089

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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 × 104 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 kdec = 112 s-1 and kred = 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 FADH2 by O2 to yield H2O2 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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570 Biowissenschaften, Biologie

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ISO 690TITTMANN, Kai, Ralph GOLBIK, Sandro GHISLA, Gerhard HÜBNER, 2000. Mechanism of Elementary Catalytic Steps of Pyruvate Oxidase from Lactobacillus plantarum. In: Biochemistry. 2000, 39(35), pp. 10747-10754. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi0004089
BibTex
@article{Tittmann2000Mecha-7417,
  year={2000},
  doi={10.1021/bi0004089},
  title={Mechanism of Elementary Catalytic Steps of Pyruvate Oxidase from Lactobacillus plantarum},
  number={35},
  volume={39},
  issn={0006-2960},
  journal={Biochemistry},
  pages={10747--10754},
  author={Tittmann, Kai and Golbik, Ralph and Ghisla, Sandro and Hübner, Gerhard}
}
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