Purification of two DD-carboxypeptidases/transpeptidases with different penicillin sensitivities from Proteus mirabilis.
Two membrane-bound enzymes of Proteus mirabilis with the dual functions of peptidoglycan DD-carboxypeptidase and transpeptidase (named DD-carboxypeptidase/transpeptidase H and L) were isolated and purified by selective solubilization with the nonionic detergent Genapol X-100, affinity chromatography on matrix-bound ampicillin, and preparative isoelectric focusing in the presence of detergent. Purified enzymes H and L were, respectively, penicillin-binding proteins 4 and 5 among seven major penicillin-binding proteins present in P. mirabilis. The enzymes differed in the following properties. Enzyme H had an Mr of 49,000; isoelectric point at pH 8.2; high sensitivity to benzylpenicillin and permanent inactivation because of high stability of the enzyme-antibiotic complex EI* (half-life 300 min); fragmentation of benzylpenicillin with formation of phenylacetylglycine during the slow decay of EI*; it functioned as an endopeptidase on peptide-crosslinked side chains of peptidoglycan. Enzyme L had an Mr of 43 000; isoelectric point at pH 5.9; low sensitivity to benzylpenicillin and low stability of EI* (half-life 7.2 min) with rapid recovery of enzyme activity; no function as an endopeptidase. The properties of enzyme L were identical with those of the single active DD-carboxypeptidase found previously in the spheroplast L-form of P. mirabilis grown in the presence of benzylpenicillin. We conclude that the partial penicillin resistance of P. mirabilis, with growth as L-form and synthesis of peptide-crosslinked peptidoglycan, depends on the continuing fuction of enzyme L as a DD-carboxypeptidase and transpeptidase in the presence of the antibiotic.
D002268:Carboxypeptidases / Q000302:isolation & purification*; D002352:Carrier Proteins; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D007525:Isoelectric Focusing; D002267:Muramoylpentapeptide Carboxypeptidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010400:Penicillin G / Q000494:pharmacology; D010406:Penicillins / Q000494:pharmacology*; D011511:Proteus / Q000201:enzymology*
D002352:Carrier Proteins; D010406:Penicillins; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G