Pyruvate Decarboxylase

Abstract

Pyruvate decarboxylase, isolated from active dry bakers' yeast, dissociates into subunits of one-half the molecular weight at alkaline pH. This has been determined by chromatography on Sephadex G-200 over the pH range 6.5 to 8.0. The conditions at which protein dissociation is observed are the same as the conditions at which the cofactors thiamine pyrophosphate and Mg2+ are released and can be separated from the protein. When the holoenzyme is reconstituted in the presence of thiamine-PP and Mg2+ the active enzyme is a dimer of the same molecular weight as the original native enzyme. When the apoenzyme, subjected to the reconstitution procedure in the absence of thiamine-PP and Mg2+, is chromatographed on Sephadex G-200 at pH 6.5 the protein is eluted at an intermediate position, suggesting a monomer-dimer equilibrium which favors the monomer. It appears that thiamine-PP in addition to its catalytic role also functions in the formation of a stable dimer which is the active holoenzyme. The presence of a subunit association step in the reconstitution process which requires thiamine-PP and Mg2+ identifies a site which may be related to the unusual requirement for excess thiamine-PP in the reconstitution process. A method for selectively staining pyruvate decarboxylase on disc electrophoresis gels with a fuchsin staining reagent is described.