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Abstract

In native carbonic anhydrase the coordinated zinc ion, which is part of the active site region, may be replaced by Co(II), with retention of enzyme activity. The Co(II)-substituted protein has spectral properties which make it more amenable to spectroscopic studies.

The protolytic reactions in the active site region of bovine Co(II)-carbonic anhydrase B have been studied by spectrophotometric titrations employing a new computer-controlled high performance titration system [1] and by temperature jump and electric field jump relaxation spectrometry. Among the fast elementary steps involved, it has been possible to differentiate between the initial protonation/deprotonation reactions, probably occurring at the surface region, an intramolecular proton transfer to the active site region and a rearrangement within the coordination sphere of the heavy metal ion.

The binding constants of the anionic inhibitors sulphate, cyanide, cyanate and thiocyanate to bovine Co(II)-carbonic anhydrase B have been determined employing the new spectrophotometric titration system. The experiments were carried out either by pH titrations at various inhibitor concentrations or by anion titrations at various pH values.

The dynamic properties of the anionic binding site of Co(II) carbonic anhydrase have been characterized on the basis of temperature jump studies. The kinetic results obtained from the investigation of the elementary steps involved in the binding of anionic inhibitors such as cyanide, cyanate and thiocyanate provide information about the mechanism of action of this enzyme. Evidence will be presented that the overall binding of monovalent anions must be characterized by a process comprising at least two steps, one corresponding to the actual binding and one corresponding to rearrangements of the anion-enzyme complex.