Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum.
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.
Tittmann
K
K
Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie and Institut für Biotechnologie, Kurt Mothes Strasse 3, D-06099 Halle, Germany.
Proske
D
D
Spinka
M
M
Ghisla
S
S
Rudolph
R
R
Hübner
G
G
Kern
G
G
D002384:Catalysis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D011771:Pyruvate Oxidase / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
D005182:Flavin-Adenine Dinucleotide; D011771:Pyruvate Oxidase; D013835:Thiamine Pyrophosphate