Analyses revealed that the native EndoT5 undergoes strong structural rearrangements as a result of Zn2+ binding. This structural rearrangement resulting in the formation of an active enzyme is completed by the Ca2+ binding. In this case, the NMR spectra uncover the tautomerism of the NH protons of histidine imidazoles responsible for the Zn2+ coordination. For the EndoT5 analogues with point substitutions in the Ca2+-binding site, similar conformational rearrangements are observed upon Zn2+ binding. However, no characteristic changes in the NMR spectra associated with the Ca2+ binding were detected. The roles of the proton exchange in the process of Ca2+-induced activation of the enzymatic activity of EndoT5 is discussed.