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|Title:||Studies on the Mechanism of the Pyruvate Oxidase Flavoprotein of Escherichia Coli (Rapid Kinetics, Biochemical Software, Lipid-Activated Enzyme)|
|Author(s):||Mather, Michael Wayne|
|Department / Program:||Chemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The pyruvate oxidase flavoprotein catalyzes the oxidative decarboxylation of pyruvate to acetate and carbon dioxide. Flavin adenine dinucleotide and thiamin pyrophosphate are cofactors. The enzymatic activity of the purfied flavoprotein is very low in the absence of lipids. Following reduction by substrate, lipids and many other other amphiphilic substances bind to a high affinity site on the flavoenzyme and stimulate the activity about 25 fold. The enzyme can also be activated by controlled proteolysis of the reduced flavoenzyme, which destroys the high affinity lipid site.
The properties of the bound flavin adenine dinucleotide were studied by spectroscopic and chemical methods. The bound flavin is inaccessible to external reagents. The visible absorption spectrum indicates that the flavin participates in hydrogen bonding but is sequestered from the bulk solvent by the protein. Upon proteolytic activation, the flavin becomes more accessible to external reagents, the spectrum changes to a form consistent with solvent contact. The flavoenzyme forms an anionic flavosemiquinoine and stabilizes the benzoquinoid forms of 6-hydroxy-flavin adenine dinucleotide and 8-mercapto-flavin adenine dinucleotide when these are substituted for the flavin adenine dinucleotide in the flavoenzyme. This suggests a polarization of the bound flavin cofactor enhancing the electrophilicity of the flavin N(5) position. However, the bound flavin cofactor does not form an adduct with the nucleophile sulfite, and the reduced flavoenzyme reacts with oxygen to form the one electron reduced superoxide radical. Consideration of these and other properties of the flavoprotein together with the known chemistry of flavins and thiamin suggests that the enzymatic reaction proceeds via free radical chemistry.
Kinetic experiments with the flavoprotein indicate that the mechanism of the enzymatic reaction is complex. At low concentrations of pyruvate, the reaction of the unactivated pyruvate oxidase flavoprotein does not appear to attain a steady state. The reductive and oxidative half cycles of the unactivated enzymatic reaction were examined in stop flow experiments and found to be quite slow. It is likely that both the reduction and oxidation of the flavin cofactor are accelerated upon activation.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.
|Date Available in IDEALS:||2014-12-15|