Studies on the Flavin Binding Properties of Native and Protease-Activated Pyruvate Oxidase From Escherichia Coli
Recny, Michael Anthony
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https://hdl.handle.net/2142/70531
Description
Title
Studies on the Flavin Binding Properties of Native and Protease-Activated Pyruvate Oxidase From Escherichia Coli
Author(s)
Recny, Michael Anthony
Issue Date
1983
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
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.
The specific activity of pyruvate oxidase may be increased more than 20 fold after limited proteolytic digestion by (alpha)-chymotrypsin in the presence of pyruvate and thiamin pyrophosphate. The "activation" phenomenon is due to the specific cleavage of a M(,r) = 2000 peptide from each subunit. The "activation peptide" ((alpha)) may be readily separated from the activated enzyme by HPLC under non-denaturing conditions. The (alpha) peptide is not required to maintain the modified tetramer in the activated state. Cleavage of the (alpha) peptide from each monomer is directly correlated with a substantial change in the visible spectrum of the flavin. The flavin shifts from a native domain characteristic of a hydrophobic pocket to one that is more hydrophilic and accessible to the aqueous environment. Proteolytic cleavage by (alpha)-chymotrypsin in the absence of thiamin pyrophosphate irreversibly inactivates the enzyme by cleavage at a different site, producing a M(,r) = 9000 "inactivation peptide" ((beta)). The (beta) peptide remains non-covalently associated with the inactivated tetramer. Cleavage of the (beta) peptide does not alter the spectrum of the flavin, even though the (beta) peptide contains the (alpha) peptide sequence. These results suggest that cleavage and release of the (alpha) peptide opens up the flavin active site and may be directly responsible for the observed stimulation of enzymatic activity. The (beta) peptide has been isolated and purified by reverse-phase HPLC, and the amino acid composition determined by acid hydrolysis.
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