Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum

Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stoppe d-flow in combination with kinetic solvent isotope effect studies. Reversib le substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M-1 s(-1) and an off-rate of pyruvate of 20 s(-1). Decarboxylation of the inter mediate lactyl-ThDP and the reduction of FAD which consists of two consecut ive single electron-transfer steps from HEThDP to FAD occur with rates of a bout k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediate s are not observed during reduction, and kinetic solvent isotope effects ar e absent, indicating that electron transfer and protonation processes are n ot rate limiting in the overall reduction process. Reoxidation of FADH(2) b y O-2 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 estim ated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate sa turation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is h ydrolyzed 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 d irectly by FAD absorbance changes using the sequential stopped-flow techniq ue.