The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and lowmolecular mass (LMM) enzymes. The latter group, which is subdivided into classes A−C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 DD-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown. T he bacterial DD-peptidases catalyze the final steps of peptidoglycan (cell wall) biosynthesis 1,2 and are efficiently inhibited by β-lactam antibiotics.3 Resistance to these antibiotics continues to emerge, particularly from evolution of new β-lactamases, enzymes that catalyze hydrolytic destruction of β-lactams, but also by the evolution and dispersal of β-lactam-resistant DD-peptidases. and Neisseria gonorrhoeae. 10Many attempts have been made to develop new β-lactams effective against mutant 11,12 but, to date, these have not been completely successful. The ideal of a broadspectrum β-lactam not susceptible to loss of effectiveness through DD-peptidase mutation has been difficult to achieve. To a considerable extent, this is likely due to the largely trial and error approach to new β-lactams. The alternative approach of targeting the reaction center and the common and essential structural features of the DD-peptidase active site has not been pursued as vigorously. C...

High molecular mass penicillin-binding proteins (PBPs, DD-peptidases) of class B, such as Streptococcus pneumoniae PBP2x, catalyze the cross-linking of peptidoglycan in bacterial cell wall biosynthesis and are thus important antibiotic targets. Despite their importance in this regard, structure-function studies of ligands of these enzymes have been impeded by the absence of useful substrates. In vitro, these enzymes do not catalyze peptide hydrolysis or aminolysis, their in vivo reaction, but some, such as PBP2x, do catalyze these reactions of certain thioesters such as PhCHCONHCHCOSCH(D-Me)CO (2). We have now prepared several peptidoglycan-mimetic thioesters that we expected to more closely resemble the natural substrates of these enzymes. To our surprise, however, these compounds, although indeed substrates of PBP2x, did not, unlike 2, appear to form an acyl-enzyme intermediate during hydrolysis, and their turnover was inhibited by certain peptides and N-acylamino acids much more weakly than that of 2. An inhibitor of this type, N-benzyloxycarbonyl-d-glutamic acid, also quenched the fluorescence of PBP2x that had been labeled at the DD-peptidase active site by 6-dansylamidopenicillanic acid. These results were interpreted in terms of a model where the peptidoglycan-mimetic thioesters preferentially bound to and hydrolyzed at a site other than the classical DD-peptidase active site. This second site is likely to represent part of an extended binding site that accommodates a peptidoglycan substrate or regulator in vivo. Such a site may be a target for future inhibitor/antibiotic design.