Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding

Sulfhydryl Chemistry Detects Three Conformations of the Lipid Binding Region of Escherichia coli Pyruvate Oxidase

“…This is consistent with the lack of cooperativity seen for the PoxB enzymatic reaction (Wang et al, 1991) (each monomer acts as a separate kinetic entity) and the lack of alterations in the global structure of PoxB (see above). The only observed cooperativity is in binding a tetramer to lipid vesicles where two functional C termini are required (Wang et al, 1991).…”

“…The 18 remaining plasmids (pYYC186-pYYC203) were constructed with the ung/dut site-directed mutagenesis method (Kunkel et al, 1987). These plasmids were all derived from plasmid pYYC102 (Wang et al, 1991) which encodes the wild type oxidase. Plasmids pYYC186-pYYC195 which encode the PoxB proteins W570C, N569C, T568C, K567C, A566C, L565C, I563C, E561C, G559C, and R558C, respectively, were constructed by use of a series of 10 50-mer oligonucleotide primers of the same nucleotide sequence (5′-TA CCT TAG CCA GTT TGT TTT CGC CAG TTC GAT CAC TTC ATC ACC GCG TCC-3′) except that a cysteine anticodon, GCA, replaced one amino acid anticodon to be mutated (underlined) in each primer.…”

“…Deletion of the C-terminal region and mutations within this region result in loss of lipid binding, indicating that the subunit C termini are the sites of lipid binding (Grabau & Cronan, 1986b;Grabau et al, 1989). In previous work, we formed tetramers composed of mixtures of normal subunits and subunits lacking the C terminus and showed that a pair of C termini is required for lipid binding (Wang et al, 1991). We subsequently used site-specific disulfide cross-linking to study the C termini in greater detail (Chang & Cronan, 1995).…”

Rate Constants in Two Dimensions of Electron Transfer between Pyruvate Oxidase, a Membrane Enzyme, and Ubiquinone (Coenzyme Q8), Its Water-Insoluble Electron Carrier

“…Therefore, in the presence of the TPP concentration used in the electrochemical experiments we carried out, the enzyme can be taken as existing permanently in its TPP complex form which will be noted E-FAD or E-FADH 2 in the following, E-FAD and E-FADH 2 being the oxidized and the reduced active enzymes, respectively. In the following, we will also consider that the surface concentration in active enzyme Γ E is the active flavin surface concentration assuming each subunit of the activated tetramer exhibits the same activity (20). Among the numerous reports concerning the activation of POx by amphiphiles, only a few dealt with the catalytic mechanism (17,18,36).…”

“…However, in the presence of substrate and TPP, the reduced enzyme undergoes a conformational change exposing a membrane binding site ( , ). Under the latter conditions, the POx structure is tetrameric, the enzyme is activated by phospholipids and manifests the catalytic properties of a true peripheral enzyme ( , ).…”