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Conformational studies of escherichia coli pyruvate oxidase
Journal article   Peer reviewed

Conformational studies of escherichia coli pyruvate oxidase

Thomas A. O'brien, Emma Shelton, Michael Mather and Robert B. Gennis
Biochimica et biophysica acta, Protein structure and molecular enzymology, v 705(3), pp 321-329
1982
PMID: 6751398

Abstract

Cofactor binding E. coli Pyruvate oxidase Quaternary structure
Pyruvate oxidase (pyruvate: oxygen oxidoreductase (phosphorylating), EC 1.2.33) 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 tbe 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 spectrun 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 after 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.

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Collaboration types
Domestic collaboration
Web of Science research areas
Biochemistry & Molecular Biology
Biophysics
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