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.
Pyruvate oxidase (POX)1 from Lactobacillus plantarum (EC 1.2.3.3) is a homotetramer. Each subunit (mass, 65.5 kDa) binds one FAD and one thiamine diphosphate (ThDP) in the presence of Mn 2ϩ or Mg 2ϩ . In the presence of oxygen and phosphate, the POX holoenzyme catalyzes the oxidative decarboxylation of pyruvate (1-3) according to the equationIn the presence of Mn 2ϩ , both coenzymes FAD and ThDP can form binary complexes with the apoenzyme that are enzymatically inactive in the native overall oxidation reaction (4).The important catalytic steps in the POX reaction are: step 1, deprotonation of C2-H of ThDP, the initial step shared by all ThDP dependent enzymes; step 2, binding of pyruvate to the C2 atom of enzyme-bound ThDP; step 3, decarboxylation of pyruvate to hydroxyethyl-ThDP; step 4, oxidation of hydroxyethylThDP by FAD; and step 5, reoxidation of reduced FAD by oxygen. To study the role of both coenzymes in POX catalysis, we have used four different approaches that selectively investigate some of the individual steps enumerated above: (a) In the initial reaction (step 1), the deprotonation of the C2-atom of the ThDP in POX holoenzyme as well as in the binary ThDP complex was studied using 1 H NMR by following the C2-H/D exchange (5). (b) DCPIP is known to accept electrons from hydroxyethyl-ThDP in other ThDP-dependent enzymes (6, 7). Here it was used as an artificial acceptor to assess the mode of electron transfer to O 2 . Two alternative donor loci have to be considered, the ThDP adduct itself or FADH 2 , to which redox equivalents could be transferred from ThDP. We have also used this assay with POX in which normal FAD was substituted with the analogue 5-deaza FAD (5-dFAD), which is essential...