Protonography and anion inhibition profile of the α-carbonic anhydrase (CruCA4) identified in the Mediterranean red coral Corallium rubrum

CruCA4 is a secreted isoform of the α-carbonic anhydrase (CA, EC 4.2.1.1) family, which has been identified in the octocoral Corallium rubrum. This enzyme is involved in the calcification process leading to the formation of the coral calcium carbonate skeleton. We report here experiments performed on the recombinant CruCA4 with the technique of protonography that can be used to detect in a simple way the enzyme activity. We have also investigated the inhibition profile of CruCA4 with one major class of CA inhibitors, the inorganic anions. A range of weak and moderate inhibitors have been identified having K_I in the range of 1–100 mM, among which the halides, pseudohalides, bicarbonate, sulfate, nitrate, nitrite, and many complex inorganic anions. Stronger inhibitors were sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethylditiocarbamate, which showed a better affinity for this enzyme, with K_I in the range of 75 μM–0.60 mM. All these anions/small molecules probably coordinate to the Zn(II) ion within the CA active site as enzyme inhibition mechanism.