Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with carboxylates

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with carboxylates including aliphatic (formate, acetate), dicarboxylic (oxalate, malonate), hydroxy/keto acids (L-lactate, L-Malate, pyruvate), tricarboxylic (citrate), or aromatic (benzoate, tetrafluorobenzoate) representatives, some of which are important intermediates in the Krebs cycle, is presented. The cytosolic isozyme hCA I was strongly activated by acetate, oxalate, pyruvate, L-lactate, and citrate (K-A around 0.1 muM), whereas formate, malonate, malate, and benzoate were weaker activators (K-A in the range 0.1-1 mM). The cytosolic isozyme hCA 11 was weakly inhibited by all the investigated anions, with inhibition constants in the range of 0.03-24 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by carboxylates, showing a K-I of 99 nM for citrate and oxalate, of 2.8 muM for malonate and of 14.5 muM for pyruvate among others. The mitochondrial isozyme hCA V was weakly inhibited by all these carboxylates (K(I)s in the range of 1.67-25.9 mM), with the best inhibitor being citrate (K-I of 1.67 mM), whereas this is the most resistant CA isozyme to pyruvate inhibition (K-I of 5.5 mM), which may be another proof that CA V is the isozyme involved in the transfer of acetyl groups from the mitochondrion to the cytosol for the provision of substrate(s) for de novo lipogenesis. Furthermore, the relative resistance of CA V to inhibition by pyruvate may be an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of this anion within this organelle. The transmembrane, tumor-associated isozyme hCA IX was similar to isozyme II in its slight inhibition by all these anions (KI in the range of 1.12-7.42 mM), except acetate. lactate. and benzoate, which showed a K-I > 150 mM. The lactate insensitivity of CA IX also represents an interesting finding, since it is presumed that this isozyme evolved in such a way as to show a high catalytic activity in hypoxic tumors rich in lactate, and suggests a possible metabolon in which CA IX participates together with the monocarboxylate/H+ co-transporter in dealing with the high amounts of lactate/H+ present in tumors. (C) 2004 Elsevier Ltd. All rights reserved.