Effect of Metal Ions on the Structure and Activity of Calcium-Activated Neutral Protease (CANP)

Abstract

To clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca²⁺), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn²⁺. Mn²⁺ significantly increases the sensitivity of CANP to Ca²⁺ but CANP is not active with Mn2+ alone. The structural changes induced by Mn²⁺ were compared with those induced by Ca²⁺, and the structure of μCANP, which is active at μM Ca²⁺, was also examined for comparison.
Mn²⁺ and Ca²⁺ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca²⁺ was not observed with Mn²⁺. Six moles of calcium bound to mCANP and the average dissociation constant of Ca²⁺ was 150 μM. The structure of μCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of μCANP indicated that the structure of μCANP was looser or SH groups were more exposed than in the case of mCANP.
A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca²⁺ can be explained in terms of conformational change and activation of the active-site SH group of CANP.