Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography.
An LD-carboxypeptidase releasing the terminal D-Ala from UDP-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala (UDP-MurNAc-tetrapeptide) was purified from Escherichia coli to biochemical homogeneity and characterized biochemically. Final purification was achieved by nocardicin A-Sepharose affinity chromatography. An apparent molecular weight of 32,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme, which seems to be a monomeric protein as indicated by gel filtration. The optimum pH of the enzyme was 8.4, and the pI was 5.5. The Km for UDP-MurNAc-tetrapeptide was 1.5 x 10(-4) M, and the Vmax was 0.4 nmol/min. Nocardicin A inhibited the enzyme competitively, with a Ki of 5 x 10(-5) M. Benzylpenicillin, cephalosporin C, thienamycin, and D-alanyl-D-alanine did not affect the enzyme activity. Possible functions of the enzyme for growth and division of the murein sacculus are discussed.
Ursinus
A
A
Abteilung Biochemie, Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany.
Steinhaus
H
H
Höltje
J V
JV
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D001667:Binding, Competitive; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D007769:Lactams
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D007769:Lactams; C027318:nocardicin; D002268:Carboxypeptidases; C021505:LD-carboxypeptidase