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Activation of Thiamin Diphosphate and FAD in the Phosphatedependent Pyruvate Oxidase fromLactobacillus plantarum

1998; Elsevier BV; Volume: 273; Issue: 21 Linguagem: Inglês

10.1074/jbc.273.21.12929

1083-351X

Kai Tittmann, Daniela Proske, Michael Spinka, Sandro Ghisla, Rainer Rudolph, Gerhard Hübner, Günther Kern,

Metabolism and Genetic Disorders

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 CO 2 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 O 2 . This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O 2 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.