Structural consequences of the binding of metal ions (regulatory Ca2+ and catalytic Zn2+) to the metalloenzyme l-alanyl-d-glutamate peptidase of the bacteriophage T5 (Endo T5) and some of its analogues containing single amino acid substitutions in the active center were analyzed by nuclear magnetic resonance (NMR), circular dichroism (CD) and calorimetry. 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.
Keywords: EndoT5; Metal-binding protein; NMR.
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