The Activities in vitro of dd ‐Carboxypeptidase and LD ‐Carboxypeptidase of Gaffkya homari during Biosynthesis of Peptidoglycan

The synthesis in vitro of peptidoglycan from the precursor UDP‐N‐acetylmuramyl‐peptide, i.e. UDP‐MurNAc‐Ala1‐dGlu2(Lys3‐dAla4‐dAla5) where the indices refer to the position of an amino acid residue within the peptide subunit, radioactively labelled in either position 3, 4 or 5 and from UDP‐N‐acetylglucosamine was studied with a wall‐membrane enzyme preparation from Gaffkya homari. The peptide subunit was partially degraded by the action of dd‐carboxypeptidase(s) and ld‐carboxypeptidase(s), resulting in the formation of polymer containing pentapeptides, tetrapeptides and tripeptides. In the wall‐bound peptidoglycan the percentage distribution of the respective peptides is 48:23:29 whereas in the soluble peptidoglycan it is 82:12:6. In the absence of concomitant peptidoglycan synthesis, i.e. in the absence of UDP‐N‐acetylglucosamine or in the presence of moenomycin, the release of d‐alanine from position 5 was only 22% of the corresponding release with concomitant peptidoglycan synthesis and, most striking, virtually no d‐alanine was released from position 4 of the peptide subunit. When UDP‐MurNAc‐tetrapeptide was used as a substrate, the degradation of the peptide subunit was catalyzed by ld‐carboxypeptidase exclusively; thus, the polymer contained tripeptides besides non‐degraded tetrapeptides. The percentage distribution of the two oligopeptides in the wall‐bound and in the soluble peptidoglycan were 37:63 and 18:82, respectively. In the absence of concomitant peptidoglycan synthesis, no d‐alanine was released from UDP‐MurNAc‐tetrapeptide. Both dd‐carboxypeptidase and ld‐carboxypeptidase act on soluble peptidoglycan with low efficiency whereas they do not act on the wall‐bound polymer. It must be assumed that the true substrate of both carboxypeptidases is the nascent peptidoglycan. This intermediate is not the soluble peptidoglycan since pulse‐chase experiments showed that this product formed in vitro is only a very poor substrate for synthesis of wall‐bound peptidoglycan.