Dipeptide binding to the extended active site of the Streptomyces R61 D-alanyl-D-alanine-peptidase: The path to a specific substrate

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-D-alanine(DD)-peptidases/transpeptidases. The natural sub strates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The bindi ng of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent beta-lactam pr obe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the beta-lactam binding site. Hydrophobic peptides su ch as Gly-L-Met and Gly-L-aminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combinati on of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-alpha-amino-epsi lon-pimelyl-D-alanyl-D-alanine Steady-state kinetic measurements establishe d this compound as the most specific peptide substrate yet discovered for a DD-peptidase by at least 3 orders of magnitude (k(cat) = 69 s(-1), K-m = 7 .9 k(cat)/K-m = 8.7 x 10(6) s(-1) M-1); acylation was rate-determining at s aturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of othe r DD-peptidases.