Detection by site-specific disulfide cross-linking of a conformational change in binding of Escherichia coli pyruvate oxidase to lipid bilayers.
Escherichia coli pyruvate oxidase, a peripheral membrane homotetrameric flavoprotein, exposes its C-terminal lipid binding site in the presence of substrate pyruvate and co-factor thiamine pyrophosphate Mg2+ and binds tightly to phospholipid bilayers during catalysis. Using site-specific disulfide cross-linking, we demonstrate that disulfide cross-links are formed between C termini of D560C pyruvate oxidase and that the degree of cross-linking is greatly increased by the presence of substrate and co-factors indicating a conformational change that results in juxtaposition of two subunit C termini. The cross-linked oxidase is enzymatically active and remains able to associate with lipid micelles. These results argue strongly that lipid bilayer binding of pyruvate oxidase involves pairing of the C termini of two subunits.
Chang
Y Y
YY
Department of Microbiology, University of Illinois, Urbana 61801, USA.
Cronan
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D007778:Lactobacillus / Q000201:enzymology; D008051:Lipid Bilayers; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011771:Pyruvate Oxidase / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003432:Cross-Linking Reagents; D017931:DNA Primers; D004220:Disulfides; D008051:Lipid Bilayers; D011771:Pyruvate Oxidase; D003545:Cysteine