Pyruvate oxidase is a peripheral membrane flavoenzyme isolated from Escherichia coli. Lipids have been shown to influence dramatically the kinetics of the enzymatic reaction; the Vmax is enhanced by about 25-fold, and the Km for both the substrate, pyruvate, and the cofactor, thiamin pyrophosphate, are altered in the presence of lipids. In addition, the Hill coefficient for thiamin pyrophosphate determined using steady-state kinetics is influenced by lipids. In this paper the interaction between thiamin pyrophosphate and pyruvate oxidase has been studied both by equilibrium dialysis and by measuring the ligand-induced quenching of the protein fluorescence. It has been shown that the enzyme possesses one thiamin pyrophosphate binding site per subunit, and that a divalent cation such as Mg2+ is required for binding. The dependence of the initial rate of the enzymatic reaction on thiamin pyrophosphate concentration is an excellent reflection of the cofactor binding. It has been demonstrated that both the average dissociation constant and the shape of the isotherm describing cofactor binding are influenced very strongly by lipids. The observed effects are different for the different lipids and detergents used in this study. The results clearly illustrate one manner in which lipids can affect the behavior of a membrane enzyme, by modulating the interaction between the enzyme and those ligands involved in catalysis.
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