Studies on the Proteolytic Activation of the Membrane Enzyme, Pyruvate Oxidase
Russell, Patricia Mcelligott
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Permalink
https://hdl.handle.net/2142/67224
Description
Title
Studies on the Proteolytic Activation of the Membrane Enzyme, Pyruvate Oxidase
Author(s)
Russell, Patricia Mcelligott
Issue Date
1980
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Pyruvate oxidase is a peripheral membrane flavoenzyme which has been purified to homogeneity from Escherichia coli. The enzyme is a tetramer of four apparently identical subunits with a molecular weight of 60,000 each. Amino acid analysis reveals that only 46% of the amino acid residues are hydrophobic. The specific activity of the enzyme is enhanced about 25-fold when assayed in the presence of some amphiphiles. A similar activation of the enzyme is observed upon controlled proteolysis. Reduction of the flavoenzyme, either by dithionite or by the simultaneous presence of substrate, pyruvate, and cofactor, thiamin pyrophosphate, is required for proteolytic activation to occur. The enzyme is proteolytically inactivated in the absence of thiamin pyrophosphate regardless of the flavin oxidation-reduction state. Activation by endopeptidases such as chymotrypsin corresponds to a reduction of the subunit molecular weight to 56,000 on sodium dodecyl sulfate polyacrylamide gels. However, sedimentation velocity studies combined with quasi-elastic light scattering results show that the proteolysis has no effect on the structure of the native tetramer. Amino-terminal analysis of native and protease-activated pyruvate oxidase indicates that it is a carboxy-terminal peptide that is being removed upon proteolysis. In fact, in the presence of pyruvate and thiamin pyrophosphate, carboxy-peptidase Y activates pyruvate oxidase, with the concomitant release of just two amino acids. Lipid activation and chymotrypsin activation of pyruvate oxidase are mutually exclusive. The presence of lipids prevents proteolytic activation as judged by the appearance of the 56,000 molecular weight band on sodium dodecyl sulfate-polyacrylamide gels. The specific activity of the protease-activated enzyme is not further increased by lipids and this form of the enzyme does not bind lipids as does the native enzyme. A structural model for pyruvate oxidase based on these findings is proposed.
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