Bromopyruvate as an affinity label for baker's yeast flavocytochrome b2. Kinetic study of the inactivation reaction.

Bromopyruvate was shown to completely inactivate cytochrome b2 in a reaction that obeyed the kinetic criteria required for affinity labels: it inactivated flavocytochrome b2 according to saturation kinetics, and the inactivation reaction was competitively inhibited by the substrate or competitive inhibitors. Inactivation was irreversible. The behaviour of both forms of flavocytochrome b2 (lintact and proteolytically cleaved) was examined. It was found that the reduced cleaved enzyme was not inactivated by bromopyruvate; this phenomenon can probably be ascribed to a structural change undergone upon reduction. The value of the lactate dissociation constant of intact cytochrome b2 cytochrome b2 was determined in competition experiments with bromopyruvate. By comparison with the divergent published values for the Ks of the cleaved from, it appears that only those that differ from the Km by a factor of two or three are reasonable. This study opens the way for the identification of an active site residue and localization in the peptide chain of the bifunctional enzyme.