Bioinformatics analysis of the sequences of orthologous zinc-containing peptidases of the M15_C subfamily revealed the presence of a conserved tryptophan residue near the active site, which is not involved in the formation of the protein core. Site-directed mutagenesis of this Trp114/109 residue using two representatives of the family, l-alanoyl-d-glutamate peptidases of bacteriophages T5 (calcium-activated EndoT5) and RB49 (EndoRB49, without ion regulation) as examples, and further analysis of the H NMR spectra of the mutants showed that a decrease in the volume of the W → F → A residue leads to changes in the hydrophobic core and active center of the protein, and also decreases the affinity for regulatory Ca in the EndoT5 mutants. The inactive T5W114A mutant lacks the ability to bind the substrate. In general, the conserved Trp114/109 residue, due to the spatial restrictions of its side chain, significantly affects the formation of the catalytically active form of the enzyme and is critical for catalysis.