Cyanogen as a selective probe for carbonic anhydrase hydrolase activity

The salt bridge probe cyanogen (ethanedinitrile, C₂N₂; N?C–-C?N) inhibits the bovine carbonic anhydrase (EC 4.2.1.1.) hydrolase activity toward various types of esters without significant effect on its hydrolyase activity. Two sets of pyridine derivatives that were isosteric substrates for the two activities were differentially affected. Acetazolamide and salamide are reversible inhibitors of the enzyme; only salamide affords protection of the hydrolase activity against the action of C₂N₂. Since each is known to bind in different positions within the active site, the selective effect of salamide may arise from its position covering one CO₂ site as well as a site important for hydrolase activity. The C₂N₂ concentration dependence of the time course of hydrolase inhibition is consistent with the existence of a high C₂N₂ affinity site with slow covalent change and a second site with lower C₂N₂ affinity, but higher rate of covalent modification of the enzyme. © 1993 John Wiley & Sons, Inc.