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 Vm.x 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.A wide range of bacterial carboxypeptidases cleaving off terminal alanine residues from the peptide substituents of murein have been described (28). Three DD-carboxypeptidase activities in Escherichia coli, are known to specifically hydrolyze the D-alanyl-D-alanine terminus of the pentapeptidyl sidechain -L-Ala-D-Glu-m-A2pm-D-Ala-D-Ala (13). Being penicillin-sensitive enzymes, they belong to the family of penicillin-binding proteins (PBPs). Whereas PBP 5 and PBP 6 are specific carboxypeptidases (29), PBP 4 has been found, in addition to its DD-carboxypeptidase activity, to hydrolyze the murein-cross-linking DD-peptide bond between D-Ala and m-A2pm, thus also showing endopeptidase activity (19).The DD-carboxypeptidases have been studied in considerable detail as a model system for the interaction of P-lactams with the active sites of penicillin-sensitive enzymes (9, 16). However, the specific function of these carboxypeptidases in the process of growth and division of the murein sacculus is still not understood. A regulatory role is indicated by their interference with the levels of pentapeptidyl residues, which represent the donors for the crucial murein cross-linking reaction catalyzed by DD-transpeptidases (14). Experimental evidence seems to support this proposal (21, 25), and interestingly mutants lacking all three DD-carboxypeptidases have not been obtained yet. Penicillin-insensitive LD-carboxypeptidases that remove the terminal D-Ala from the tetrapeptidyl moiety -L-Ala-DGlu-m-A2pm-D-Ala have also been identified in E. coli (1,15,23,24). One LD-carboxypeptidase present in E. coli has been purified and determined to be a monomeric protein with a molecular mass of 12 kDa (24). Another LD-carboxypeptidase has been partially purified and reported to be an 86-kDa protein consisting of two 43-kDa polypeptides (2). This enzyme can easily be extracted from cells by Tris-EDTA, suggesting a localization in the periplasm (1). In synchronized cultures, the enzyme was found to be most active at the time of cell division. The activity of the enzyme has been proposed to be controlled by a still unidentified inhibitor (2).Re...