Abstract
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.
Highlights
Activity might have been due to variations in the quantity of this substrate. In this communication we report the purification and characterization of the enzyme carboxypeptidase-IIW
This enzyme activity is different from the diaminopimelateincorporating activity in the membrane particles of B. megaterium KM as described by Wickus and Strominger [1, 2]
For the GlcNAc moiety could explain in part the high affinity of this enzyme for the cell wall. This enzyme fails to utilize walls derived from other organisms that contain meso-diaminopimelate in their peptidoglycan and have the disaccharide-tetrapeptide of composition identical with that of B. megaterium as one of their wall components
Summary
The direct assay used the same assay incubation and analysis except the final volume was increased from 20 ~1 and the enzyme was added directly to the reaction mixture without the prior binding step. Paper Chromatography-Reaction or incubation mixtures were applied to Whatman No 3MM paper and chromatographed in isobutyric acid: 1 N NH,OH (5:3 v/v) for 18 h, unless stated otherwise. In this system the RF values of the following compounds are: peptidoglycan, 0.0 [9]; UDP-MurNAc-pentapeptide, UDP-MurNAc-tetrapeptide, and MurNAc-tetrapeptide, 0.5 [9]; bis(disaccharide-peptide). Document No 78-1396, cite author(s), and include a check or money order for $1.80 per set of photocopies
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