Importance of the two tryptophan residues in the Streptomyces R61 exocellular DD-peptidase

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp²⁷¹ was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp²⁷¹ residue, though almost invariant among the β-lactamases of classes A and C and the low-M(r) penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp²³³ into Leu and Ser strongly decreased the enzymic activity, the affinity for β-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant peptidase thus behaved as a weak β-lactamase.