Removal of Metal from Carboxypeptidase A Proceeds via a Split Pathway: Implications for the General Mechanisms of Metalloenzyme Inactivation by Chelating Agents.

Abstract

The chelation of protein-bound metal ions is typically thought to follow either a dissociative (D) or an associative (A) path. While the former mechanism involves the spontaneous dissociation of the metal from the protein prior to chelation, the latter route is characterized by the formation of an intermediate protein-metal-chelator ternary complex. Using the prototypical zinc protease carboxypeptidase A (CPA) and a variety of charged and neutral chelating agents, we demonstrate that inactivation of the enzyme (and likely other metalloproteins) proceeds through a split pathway with contributions from both D- and A-type mechanisms. In the case of charged chelators such as ethylenediaminetetraacetic acid (EDTA), the proportions of both paths could be tuned over a wide range through variation of the chelator concentration and the ionic strength, I (from ∼95% A type at low I values to ∼5% at high I values). By measuring the EDTA concentration and time dependence of CPA inactivation and fitting the obtained kinetic data to a modified time-dependent inhibition model, we obtained the rate constants for the A and D paths (k_inact and k_off, respectively) and the inhibition constant (K_I) for the formation of the CPA-Zn²⁺-EDTA ternary complex, indicating that the decreased contribution of the A-type mechanism at high ionic strengths originates from a large (40-fold; at I = 0.5 M) increase in K_I. This observation might be related to a triarginine motif in CPA that electrostatically steers negatively charged substrates into the active site and may therefore also guide carboxylate-bearing chelators toward the Zn²⁺ ion.