Studies on the Flavin Binding Properties of Native and Protease-Activated Pyruvate Oxidase From Escherichia Coli

Pyruvate oxidase, a tetrameric enzyme consisting of four identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically-active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The kinetics of the reconstitution reaction indicate that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Recny, Michael Anthony

• Chemistry, Biochemistry

Identifier

(UMI)AAI8410029

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/70531