Purification and inhibition studies with anions and sulfonamides of an a-carbonic anhydrase from the Antarctic seal Leptonychotes weddellii

A high activity a-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from various tissues of the Antarctic seal Leptonychotes weddellii. The new enzyme, denominated lwCA, has a catalytic acitivity for the physiologic CO2 hydration to bicarbonate reaction, similar to that of the high activity human isoform hCA II, with a kcat of 1.1x10E6 s-1, and a kcat/Km of 1.4 x 10E8 M-1 s-1. The enzyme was highly inhibited by cyanate, thiocyanate, cyanide, bicarbonate, carbonate, as well as sulfamide, sulfamate, phenylboronic/phenylarsonic acids (KIs in the range of 46-100 uM). Many clinically used sulfonamides, such as acetazolamide, methazolamide, dorzolamide, brinzolamide and benzolamide were low nanomolar inhibitors, with KIs in the range of 5.7 – 67 nM. Dichlorophenamide, zonisamide, saccharin and hydrochlorothiazide were weaker inhibitors, with KIs in the range of 513 – 5390 nM. The inhibition profile with anions and sulfonamides of the seal enzyme was rather different from those of the human isoforms hCA I and II. The high sensitivity to bicarbonate inhibition of lwCA, unlike that of the human enzymes, may reflect an evolutionary adaptation to the deep water, high CO2 partial pressure and hypoxic conditions in which Weddell seals spend much of their life.