Spectroscopic studies of pyruvate oxidase flavoprotein from Escherichia coli trapped in the lipid-activated form by cross-linking.
Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2. The maximal turnover of the enzyme, measured using a ferricyanide reductase assay, is increased 20-to 30-fold by either of two methods. Proteolysis in the presence of the substrate (pyruvate) and cofactor (Mg2+-thiamin pyrophosphate) results in cleavage at a single locus near the carboxyl terminus and concomitant activation. Phospholipids and detergents can bind to the enzyme and result in a similar activation, which is presumed to be physiologically relevant, since the enzyme functions as a peripheral membrane enzyme. Previous studies showed that proteolytic activation of pyruvate oxidase results in substantial changes in the absorption spectrum of the oxidized form of the bound flavin. Up to this time, similar studies of the lipid-activated form of the enzyme have not been feasible, since it is necessary to reduce the flavoprotein in order to induce binding to the lipids. In this paper, glutaraldehyde cross-linking of the lipid-activated enzyme is used to trap the enzyme in this form. Spectroscopic studies show alterations of the flavin spectrum similar to those observed upon proteolytic activation. This alteration in the flavin binding site is consistent with kinetic studies which suggest that activation results from an acceleration in the rates of electron transfer both into and out of the bound flavin, which appears to be more "accessible" in the activated forms of the enzyme.
D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005420:Flavoproteins / Q000378:metabolism*; D005976:Glutaral / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism; D011771:Pyruvate Oxidase / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013053:Spectrophotometry; D013835:Thiamine Pyrophosphate / Q000494:pharmacology
D003432:Cross-Linking Reagents; D005420:Flavoproteins; D012967:Sodium Dodecyl Sulfate; D011771:Pyruvate Oxidase; D010447:Peptide Hydrolases; D013835:Thiamine Pyrophosphate; D005976:Glutaral