Isolation and characterization of the protease-activated form of pyruvate oxidase. Evidence for a conformational change in the environment of the flavin prosthetic group.
Pyruvate oxidase is a tetrameric enzyme consisting of four identical subunits. The specific activity of the enzyme may be increased more than 20-fold by limited proteolytic digestion by alpha-chymotrypsin in the presence of pyruvate and thiamin pyrophosphate. This "activation" phenomenon is due to the specific cleavage of an Mr = approximately 2000 peptide from each subunit. The Mr = 2000 "activation peptide" (alpha) may be readily separated from the activated enzyme by high performance liquid chromatography under nondenaturing conditions. The alpha peptide is not required to maintain the modified tetramer in the activated state. Cleavage of the alpha peptide from each monomer is directly correlated with a substantial change in the visible spectrum of the flavin, characteristic of a shift from a hydrophobic to a more hydrophilic environment. Proteolytic cleavage by alpha-chymotrypsin in the absence of thiamin pyrophosphate irreversibly inactivates the enzyme by cleavage at a different site, producing an Mr = approximately 9000 "inactivation peptide" (beta). The beta peptide remains noncovalently associated with the inactivated tetramer. Cleavage of the beta peptide does not alter the spectrum of the flavin, even though the beta peptide contains the alpha peptide sequence. These results suggest that cleavage and release of the alpha peptide opens up the flavin active site and may be directly responsible for the observed stimulation of enzymatic activity.
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli; D005415:Flavins; D046911:Macromolecular Substances; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D011771:Pyruvate Oxidase / Q000302:isolation & purification* / Q000378:metabolism
D005415:Flavins; D046911:Macromolecular Substances; D011771:Pyruvate Oxidase; D010447:Peptide Hydrolases