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Soluble lytic transglycosylase 70 (Slt70), one of the better characterized murein hydrolases of Escherichia coli, was covalently bound to CNBr-activated Sepharose and used as a specific tool to screen for proteins showing an affinity for Slt70. Several proteins were specifically enriched by Slt-Sepharose affinity chromatography. Two of them were identified as the penicillin-binding proteins (PBP)3 and PBP7/8. Thus, the bifunctional synthase PBP3, specifically involved in septum formation, and PBP7/8, recently shown to be a DD-endopeptidase, bind to Slt70 in vitro. In addition, PBP7/8 was found not only to stabilize but also to stimulate the enzymatic activity of Slt70 by a protein-protein interaction. It is concluded that Slt70, PBP7/8, and PBP3 may form a multienzyme complex in vivo.

The enzyme carboxypeptidase-IIW of Bacillus megaterium incorporates free diaminopimelate into purified bacterial walls. This enzyme can be solubilized from toluene-treated cells by LiCl extraction and has now been purified 106-fold to one major band on polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of approximately 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Sephadex G-100 gel filtration. Carboxypeptidase-IIW requires divalent cations and thiol group(s) for optimal activity. Product analysis indicates that the enzyme can hydrolyze the terminal D-alanine from the tetrapeptide of the peptidoglycan or replace it with a variety of amino acids with D-asymmetric centers for transpeptidation. Substrate specificity studies reveal that the enzymatic activity depends on the presence of N-acetyl-D-glucosamine of the GlcNAc-MurNAc-tetrapeptide. This specificity of carboxypeptidase-IIW for the N-acetyl-D-glucosamine explains in part the affinity of the enzyme for the cell wall of B. megaterium. The enzyme is compared to the carboxypeptidases-transpeptidases of other organisms with the similarities and differences discussed.

Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,000; Mr = 100,000 doublet; and Mr = 46,000. The low molecular weight protein bound penicillin reversibly and was purified by prebinding membranes with penicillin prior to affinity chromatography. The purified protein catalyzed transpeptidase and carboxypeptidase reactions using di[14C]acetyl-L-lysyl-D-alanyl-D-alanine as the substrate and glycine and hydroxylamine as the acceptors. In addition, the enzyme catalyzed a penicillinase reaction. Kinetic analysis of these reactions revealed similar Vmax values suggesting that, if there is a single active site, the rate-determining steps (i.e. deacetylation) are similar. Rapid denaturation of the enzyme.substrate complex resulted in the detection of covalent penicilloyl- and diacetyl-L-lysyl-D-alanyl.enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.