Kinetics of Formation and Dissociation of Metallocarboxypeptidases

Abstract

The rates of formation of a number of metallocarboxypeptidases from metal ions and bovine apocarboxypeptidase A (CPA) have been measured directly and by a competitive method. Rates were determined within pH = 6–8 by utilising the pH change attending metal-ion incorporation, employing indicator and stopped-flow. Second-order rate constants (k_f, M⁻¹ s⁻¹ at 25°C, I = 1 M NaCl, pH = 7, Tris = 25 μM) were 1.7 × 10⁵ (Mn²⁺), 3 × 10⁴ (Co²⁺), 5 × 10³ (Ni²⁺), 7 × 10⁵ (Zn²⁺), and 9 × 10⁵ (Cd²⁺). Relative incorporation rate constants were determined at 25°, pH = 7.0, Tris = 0.1 M, by competing two metal ions for a deficiency of apoprotein and analyzing the products by differential enzyme activity. Agreement between the two methods was reasonable. Rate constants for dissociation of CoCPA, NiCPA, and ZnCPA were measured by loss of enzyme activity on addition of the metal ion scavenger EDTA. Values of k_d at 25°, I = 1.0 M NaCl, pH = 7.0 were 8 × 10⁻³, 3 × 10⁻⁵, and 4 × 10⁻⁴ s⁻¹, respectively, Values of K obtained kinetically (k_f/k_d) were in good agreement with those determined by activity measurements of equilibrated solutions. Results are compared with those of bovine apocarbonic anhydrase, where generally significantly slower rates are encountered.