PMID:6751398
| Citation |
O'Brien, TA, Shelton, E, Mather, M and Gennis, RB (1982) Conformational studies of Escherichia coli pyruvate oxidase. Biochim. Biophys. Acta 705:321-9 |
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| Abstract |
Pyruvate oxidase (pyruvate:oxygen oxidoreductase (phosphorylating), EC 1.2.3.3) is a peripheral membrane enzyme from Escherichia coli which utilizes the cofactors thiamin pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) to catalyze the decarboxylation of pyruvate to acetic acid and carbon dioxide. The specific activity of the oxidase is enhanced 25-fold when assayed in the presence of certain lipids and detergents. Previous studies have demonstrated that the affinity of pyruvate oxidase for phospholipids and detergents is substantially increased when the flavin is reduced. In this paper, several techniques are utilized to probe both the nature of the active site and the conformational changes in the protein which are concomitant with flavin reduction and with the binding of lipids to the enzyme. Analysis of the circular dichroism spectrum in the far ultraviolet region indicates that neither the binding of lipid activators to the oxidase nor reduction of the enzyme-bound flavin by pyruvate has a significant effect on the average secondary structure of the enzyme. High-resolution electron microscopy demonstrates that at low enzyme concentrations, i.e., assay conditions, incubation of the reduced flavoprotein in the presence of an amphiphilic activator does not alter the quaternary structure of pyruvate oxidase. The results indicate that the conformational changes in the protein due either to reduction of the flavin or to the binding of lipid activators are localized. |
| Links | |
| Keywords |
Circular Dichroism; Escherichia coli/enzymology; Kinetics; Microscopy, Electron; Protein Conformation; Pyruvate Oxidase/metabolism; Spectrometry, Fluorescence; Spectrophotometry; Structure-Activity Relationship |
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