Studies on the quaternary structure of Escherichia coli pyruvate oxidase

• Published in: The Journal of biological chemistry Vol. 255; no. 2; pp. 379 - 383
• Main Authors: Stevens, D.J., Gennis, R.B.
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25.01.1980
• Subjects: Azides; Cell Membrane - enzymology; Escherichia coli - enzymology; Macromolecular Substances; Naphthalenesulfonates; Protein Binding; Protein Conformation; Pyruvate Oxidase; Spectrometry, Fluorescence; Spectrophotometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)86183-5

Pyruvate oxidase is a peripheral membrane enzyme isolated from Escherichia coli. The enzyme catalyzes the oxidative decarboxylation of pyruvate to yield acetate plus CO2. The specific activity of the purified oxidase is stimulated 25-fold by lipids, and this lipid requirement has been the subject of previous studies. Since the enzyme is a tetramer at high protein concentrations (1 mg/ml) and is known to self-aggregate under certain conditions, the question arose as to whether the lipid stimulation observed in the steady state assay might be due to a change in the quaternary structure of the protein, either a dissociation or further association. This report is directed at determining the state of association of pyruvate oxidase under assay conditions by using fluorescence polarization. A photoreactive, nonspecific probe, 1-azidonaphthalene 5-sulfonate, was used to label the protein surface with an extrinsic fluorophore. It is concluded that under steady state assay conditions the oxidase remains tetrameric.