Metal ions and clostridiopeptidase A

Abstract

Clostridiopeptidase A is a collagenolytic enzyme produced by the bacterium Clostridium histolyticum. It contains four identical sub-units, each with a molecular weight of about 25,000. The enzyme contains Zn and requires Ca 2+ for activity [1]. We are using lanthanide ions (Ln 3+) and Co 2+ to investigate the role of Ca 2+ and Zn, respectively, in the catalytic activity of clostridiopeptidase A. For this purpose, it is convenient to use an artificial pentapeptide (‘Pz-peptide’) as the substrate in place of collagen whose properties are altered in the presence of Ln 3+ [2]. Previous kinetic analyses, conducted in this laboratory, of the effect of Ln 3+ on the hydrolysis of Pz-peptide by clostridiopeptidase A have suggested that Ca 2+ is required for the binding of enzyme and substrate [3]. Sm 3+ lowers the K m nearly 15 fold, but it also lowers the V max by similar amount, suggesting that the enzyme-substrate complex is an abortive one. The relative ability of different Ln 3+ to inhibit clostridiopeptidase A is Lu < Er < Sm ⪢ La. As the radius of La 3+ (1.016 Å) exceeds that of Ca 2+ (0.990 Å), while Sm 3+ (0.964 Å), Er 3+ (0.881 Å) and Lu 3+ (0.850 Å) are smaller, the Ca 2+-binding site on the enzyme appears to be sterically constrained, such that ions of greater radius than that of Ca 2+ have restricted access. Heat inactivation studies also revealed a thermostability role for Ca 2+ [3]. The ability of Ln 3+ to inhibit clostridiopeptidase A while enhancing its substrate-binding, suggests a role for these cations in the purification of this enzyme by affinity chromatography; previous attempts to do this have been foiled by collagenolysis, even at 4 °C [4]. Experiments to investigate this possibility have qualitatively confirmed the conclusions from the kinetic analyses mentioned in the preceding paragraph. When a Ca 2+-free solution of clostridiopeptidase A was passed through an affinity column packed with calf skin collagen trapped within particles of polyacrylamide [4], none of the enzyme stuck to the column. Addition of increasing amounts of Sm 3+ progressively enhanced binding, with complete sequestration of clostridiopeptidase A occuring at a Sm 3+ concentration of 75 μM. Comparison of the efficiencies of Lu 3+, Er 3+, Sm 3+ and La 3+ at a concentration of 75 μM (Table I), gave results in complete accord with those of the kinetic studies. Co 2+ enhanced the rate of hydrolysis of peptide by clostridiopeptidase A, a maximum stimulation of 2.5–3 fold being obtained with 2.5 mM Co 2+. Lineweaver-Burk analysis of this stimulation revealed an elevation of both the V max and K m by a factor of about 2.5 in the presence of 2.5 mM Co 2+. Taken at face value, these findings indicate that Co 2+ increased the catalytic efficiency of the enzyme, despite reducing its affinity for the substrate. If Co 2+ specifically replaced Zn, as happens with many other enzymes, the data suggest a previously unrecognized role for Zn in substrate-binding. However, we are also investigating possible alternative explanations, such as an action of Co 2+ at the Ca 2+-binding site of the enzyme, or its interaction with the substrate. Both Ln 3+ and Co 2+ provide useful spectroscopic properties which should greatly aid further investigation of these matters.