Regulation of carboxypeptidase E: Effect of Ca²⁺ on enzyme activity and stability

Carboxypeptidase E (CPE), an enzyme that functions in the post-translational processing of bioactive peptides, is a member of the metallocarboxypeptidase gene family. A 12-residue region of CPE has 70% amino acid identity with the bacterial enzyme carboxypeptidase T (CPT); in CPT, this region has been identified previously as the Ca²⁺-binding region (Teplyakov, A., Polyakov, K., Obmolova, G., Strokopytov, B., Kuranova, I., Osterman, A., Grishin, N., Smulevitch, S., Zagnitko, O., Galperina, O., Matz, M., and Stepanov, V. (1992) Eur. J. Biochem. 208, 281-288). Using ⁴⁵Ca²⁺ binding, we determined that CPE binds Ca²⁺. To investigate the potential function for the interaction of CPE with Ca²⁺, we investigated the effect of Ca²⁺ on aggregation, thermostability, and enzyme activity of CPE. CPE does not aggregate under a variety of Ca²⁺ concentrations at either pH 5.5 or 7.5, and with protein concentrations ranging from 10 to 100 μg/ml. Whereas Ca²⁺ generally stabilizes proteins to thermal denaturation, CPE was destabilized by Ca²⁺ and stabilized by low concentrations of EGTA. The Ca²⁺-induced destabilization of CPE was more pronounced at pH 8 than at lower pH values. At pH 8, CPE was unstable even at 37 °C, with approximately 40% loss of activity upon incubation for 30 min in the absence of added Ca²⁺ and 70% loss of activity upon incubation in the presence of 10 mM CaCl₂. Enzyme activity was not influenced by added Ca²⁺, but was stimulated by micromolar concentrations of EGTA; kinetic analysis showed this stimulation to be due to a change in V_max, and not K_m. Taken together, these data suggest that Ca²⁺ plays a role in the regulation of CPE activity.