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 Ca(2+)-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 micrograms/ml. Whereas Ca2+ generally stabilizes proteins to thermal denaturation, CPE was destabilized by Ca2+ and stabilized by low concentrations of EGTA. The Ca(2+)-induced destabilization of CPE was more pronounced at pH 8 than at lower pH values. At pH 8, CPE was unstable even at 37 degrees 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 Vmax, and not Km. Taken together, these data suggest that Ca2+ plays a role in the regulation of CPE activity.
Nalamachu
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SR
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Song
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Fricker
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LD
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology*; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D010902:Pituitary Gland / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D001426:Bacterial Proteins; D010618:Phenanthrolines; D004492:Edetic Acid; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; C076614:CPT protein, Thermoactinomyces vulgaris; D002118:Calcium