ACTIVATION OF THIAMIN DIPHOSPHATE AND FAD IN THE PHOSPHATE-DEPENDENT PYRUVATE OXIDASE FROM LACTOBACILLUS-PLANTARUM

The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillu s 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 an d CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety, The rate constant of the initi al 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 catalyti c rate constant to 20 s(-1) requires enzyme-bound FAD, FAD appears to bind in a two-step mechanism. The primarily bound form allows formatio n of hydroxyethylthiamine diphosphate but not the transfer of electron s from this intermediate to O-2, This intermediate form can be mimicke d using 5-deaza-FAD, which is inactive toward O-2 but active in an ass ay using 2,6-dichlorophenolindophenol as electron acceptor. This analo gue also promotes the rate constant of C2-H dissociation of thiamine d iphosphate in pyruvate oxidase beyond the overall enzyme turnover. For mation of the catalytically competent FAD-thiamine-pyruvate oxidase te rnary complex requires a second step, which was detected at low temper ature.