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 microM), whereas
formate,
malonate,
malate, and
benzoate were weaker activators (K(A) in the range 0.1-1mM). The cytosolic
isozyme hCA II 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 microM for
malonate and of 14.5 microM 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 5.5mM), 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 (K(I) 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.