Lipid activation and protease activation of pyruvate oxidase. Evidence suggesting a common site of interaction on the protein.

The specific activity of purified pyruvate oxidase can be increased by about 25-fold either by the binding of certain lipids or detergents to the protein, or, alternatively, by a proteolytic modification of the enzyme performed in the presence of both the substrate and cofactor. The conformational change in this peripheral membrane enzyme which is concomitant with lipid or membrane binding is apparently similar to that which results from the proteolytic cleavage which also results in activation. Previous work has demonstrated that upon proteolytic activation the subunit molecular weight of the enzyme is reduced from 60,000 to 56,000 and it is evident that in the simultaneous presence of both pyruvate and thiamin pyrophosphate a single specific region or loop of the protein becomes highly susceptible toward proteolytic attack. It has also been previously demonstrated that the lipid binding properties of pyruvate oxidase are strikingly enhanced when pyruvate oxidase is in the presence of the substrate plus the cofactor. In this paper, the relationship between the lipid-binding sites and the sites of proteolytic cleavage is explored. It is demonstrated that the affinity for lipids and detergents of the protease-activated form of the oxidase is greatly reduced compared to that of the native enzyme. Furthermore, in the presence of those lipids and detergents which activate the enzyme, pyruvate oxidase is protected against proteolysis under conditions where proteolytic activation would normally proceed. These data suggest that the site of proteolytic cleavage is directly responsible for the lipid-binding properties of this enzyme. The accessibility of this site either to lipids or to proteases is influenced by the ligands bound to the enzyme and the oxidation-reduction state of the tightly bound flavin.