Anion inhibition studies of two α-carbonic anhydrases from Lotus japonicus, LjCAA1 and LjCAA2.

The model organism for the investigation of symbiotic nitrogen fixation in legumes Lotus japonicus encodes two carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-class, LjCAA1 and LjCAA2. Here we report the kinetic characterization and inhibition of these two CAs with inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, and phenylboronic/arsonic acids. LjCAA1 showed a high catalytic activity for the CO2 hydration reaction, with a k(cat) of 7.4∗10(5) s(-1) and a k(cat)/K(m) of 9.6∗10(7) M(-1) s(-1) and was inhibited in the low micromolar range by N,N-diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic/arsonic acid (K(I)s of 4-62 μM). LjCAA2 showed a moderate catalytic activity for the physiologic reaction, with a k(cat) of 4.0∗10(5) s(-1) and a k(cat)/K(m) of 4.9∗10(7) M(-1) s(-1). The same anions mentioned above for the inhibition of LjCAA1 showed the best activity against LjCAA2 (K(I)s of 7-29 μM). Nitrate and nitrite, anions involved in nitrogen fixation, showed lower affinity for the two enzymes, with inhibition constants in the range of 3.7-7.0 mM. Halides and sulfate also behaved in a distinct manner towards the two enzymes investigated here. As LjCAA1/2 participate in the pH regulation processes and CO2 metabolism within the nitrogen-fixing nodules of the plant, our studies may shed some light regarding these complex biochemical processes.