Purification and properties of penicillin-binding proteins 5 and 6 from Escherichia coli membranes.
Penicillin-binding proteins (PBPs) 5 and 6 in the cytoplasmic membranes of Escherichia coli K12, which had previously been co-purified as a penicillin-sensitive D-alanine carboxypeptidase IA (Tamura, T., Imae, Y., and Strominger, J. L. (1976) J. Biol. Chem. 251, 414-423), were each purified to protein homogeneity. Purification involved selective solubilization of PBPs 1a, 5, and 6 from membranes by Triton X-100 at low ionic strength, covalent penicillin affinity chromatography, and CM-cellulose column chromatography. Purified PBP 5 and PBP 6 each catalyzed a D-alanine carboxypeptidase I activity using various natural and synthetic substrates including linear uncross-linked peptidoglycan. PBP 5 showed 3- to 4-fold higher specific activities toward these substrates than PBP 6. Both PBPs also catalyzed a model transpeptidase activity using glycine as a transpeptidation acceptor, and showed similar pH profiles and MgCl2 sensitivities for their D-alanine carboxypeptidase I activities. Both PBPs bound a stoichiometric amount of [14C]penicillin G at saturation. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after partial proteolysis by proteases and cyanogen bromide demonstrated that these PBPs are distinct polypeptides.
D000409:Alanine / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins; D002268:Carboxypeptidases / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005998:Glycine / Q000494:pharmacology; D006602:Hexosyltransferases; D007700:Kinetics; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010458:Peptidyl Transferases; D013379:Substrate Specificity
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010446:Peptide Fragments; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; C019632:alanine carboxypeptidase; D002267:Muramoylpentapeptide Carboxypeptidase; D000409:Alanine; D005998:Glycine