Pyruvate oxidase is a peripheral membrane flavoenzyme isolated from Escherichia coli. The in vitro specific activity of the pure enzyme is enhanced 25-fold in the presence of certain lipids and detergents. In addition, the affinity of the protein for both phospholipids and detergents is significantly enhanced in the presence of the enzyme substrate and cofactor, pyruvate and thiamin pyrophosphate (Mg2+). In this paper a novel fluorescent probe is used to examine the protein conformational changes concomitant with substrate reduction of the flavin, activation of the oxidase, and binding of the detergent activator, dodecyl sulfate. In the presence of dodecyl sulfate, the probe bis(8-p-toluidino-1-naphthalenesulfonate) (bis-Tns) binds to pyruvate oxidase with a resultant large increase in probe fluorescence at dye concentrations below 1 μM. No binding at low dye concentration is observed in the absence of the activator. The enzyme binds about 16 molecules of bis-Tns, in what appear to be hydrophobic binding sites. The substrate-reduced flavoprotein binds to bis-Tns not only.
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