Interactions of tervalent lanthanide ions with bacterial collagenase (clostridiopeptidase A)

Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm³⁺, whose ionic radius is closest to that of Ca²⁺, is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100μm in the presence of 5mm-Ca²⁺. Er³⁺ and Lu³⁺, which both have ionic radii smaller than either Ca²⁺ or Sm³⁺, inhibit less efficiently, and La³⁺, which is slightly larger than Ca²⁺ or Sm³⁺, inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca²⁺-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca²⁺ [ionic radius 0.099nm (0.99 Ȧ)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca²⁺, requires lanthanide concentrations 50–100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50μm, Sm³⁺ lowers the apparent K_m for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent V_max. from 0.29 Wünsch–Heidrich unit to 0.018 unit. Thus Sm³⁺ enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme–Sm³⁺–substrate complex. Inhibition by Sm³⁺ is competitive with regard to Ca²⁺. The apparent dissociation constant, K_d, of Ca²⁺ is 0.27mm, where the K_i for Sm³⁺ is 12μm. Clostridiopeptidase is more thermolabile in the absence of Ca²⁺. With Sm³⁺, thermoinactivation of the enzyme at 53°C or 60°C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca²⁺ at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.