Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding.
Pyruvate oxidase of Escherichia coli, an enzyme greatly activated by phospholipids, is a tetramer of a Mr 62,000 subunit. We have utilized the differing electrophoretic mobilities of several mutant oxidases on native polyacrylamide gels to study the role of the quaternary structure of the enzyme in the activation process. We found that when two poxB gene alleles coexisted in cells, heterotetrameric species were formed in addition to homotetramers. The concentration of each tetrameric species varied according to the concentration of the different subunits present, and the distribution seemed virtually identical to those expected from random mixing. We showed that the intrinsic activity of pyruvate oxidase was not affected by interactions among the four subunits. However, binding of the enzyme to lipids, a property required for function in vivo, required that a tetramer contain at least two subunits capable of lipid binding. Our data fit the model proposed previously (Grabau, C., Chang, Y.-Y., and Cronan, J. E., Jr. (1989) J. Biol. Chem. 264, 12510-12519) in which the carboxyl termini of two subunits interact to form a functional lipid-binding domain. We also have detected oxidase activity in a form of oxidase of unusually high electrophoretic mobility. This form seems to be either a monomeric or a dimeric form (more probably the former) of the oxidase subunit.
Wang
A Y
AY
Department of Microbiology, University of Illinois, Urbana 61801.
Chang
Y Y
YY
Cronan
J E
JE
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D010957:Plasmids; D011771:Pyruvate Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D010743:Phospholipids; D011771:Pyruvate Oxidase