The zinc form of carnosine dipeptidase 2 (CN2) has dipeptidase activity but its substrate specificity is different from that of the manganese form

Abstract

Carnosine dipeptidase II (CN2), a metallopeptidase present in the cytosol of various vertebrate tissues, catalyzes the hydrolysis of carnosine and several other dipeptides in the presence of Mn²⁺. Although the metal-binding center of mouse CN2 is also able to associate with Zn²⁺in vitro, it was not known whether the zinc form of CN2 has any enzymatic activity. In the present study, we show that Zn²⁺ has a higher affinity for binding to CN2 than Mn²⁺, as evidenced by native mass spectrometry. The issue of whether the zinc form of CN2 has enzymatic activity was also examined using various dipeptides as substrates. The findings indicate that the zinc form of CN2 catalyzes the hydrolysis of several different dipeptides including Leu-His, Met-His and Ala-His at a reaction rate comparable to that for its manganese form. On the other hand, the zinc form of CN2 did not catalyze the hydrolysis of carnosine and several other dipeptides that are hydrolyzed by the manganese form of CN2. Substrate specificity was also examined in HEK293T cells expressing CN2, and the findings indicate that Leu-His, Met-His, but not carnosine, were hydrolyzed in the cell culture. These results suggest that the zinc form of CN2 is an active enzyme, but with a different substrate specificity from that of the manganese form.

Keywords: CN2; CNDP2; Dipeptide; Metalloprotease; Native mass spectrometry; Substrate specificity.