High affinity lipid binding sites on the peripheral membrane enzyme pyruvate oxidase. Specific ligand effects on detergent binding.

Pyruvate oxidase is a peripheral membrane enzyme which has been purified to homogeneity from Escherichia coli. When assayed in the presence of some amphiphiles the enzymatic activity is stimulated nearly 25fold; these amphiphiles include phospholipids, neutral detergents, and charged detergents. Both anionic and cationic detergents, at concentrations well below their critical micelle concentrations, are capable of activating the enzyme. In this paper the interaction between sodium dodecyl sulfate (SDS) and pyruvate oxidase has been studied by equilibrium dialysis. It is demonstrated that pyruvate oxidase possesses a small number of high affinity detergent binding sites similar to those reported for serum apolipoproteins and that these sites are functionally significant. The primary mode of interaction between the detergent and the enzyme is hydrophobic and not electrostatic. It is also shown that the SDS-binding isotherm is strongly affected by specific ligands bound to the enzyme. The simultaneous presence of both the substrate, pyruvate, and the cofactor, thiamin pyrophosphate, results in a large increase of the affinity of the oxidase for SDS at detergent concentrations which elicit activation. The presence of either pyruvate or thiamin pyrophosphate alone has little effect on detergent binding. Fluoropyruvate, a competitive inhibitor of pyruvate oxidase, can be substituted for pyruvate and has a similar effect on the SDS-binding isotherm in the presence of thiamin pyrophosphate. It is clear that there is a ligandinduced conformational change in pyruvate oxidase which has dramatic effects on the properties of the enzyme. This conformational change results in a great enhancement of the detergent-protein interactions which are observed.