Fig. 1: Structure of native carbonic anhydrase II (Zn-CA II) and its catalytic mechanism.

a The active site consists of zinc binding site, hydrophobic/hydrophilic regions, and entrance conduit (EC). b The water networks in the active site are responsible for the proton transfer (red) and substrate/product/water exchange (blue) during enzyme catalysis. c The CO₂ hydration reaction mechanism of Zn-CA II. First, CO₂ binds to the active site, leading to a nucleophilic attack by the zinc-bound hydroxyl ion onto CO₂. HCO₃⁻ thus formed is subsequently displaced by the water molecule inflowing through EC. The HCO₃⁻ molecule likely binds to Zn²⁺ ion in a monodentate mode and its OH group is held at the Zn²⁺ ion due to the hydrogen bonding with Thr199^{52, 53}. This product binding configuration leads to a weak interaction between the product and Zn²⁺ ion, thereby facilitating fast product dissociation⁵⁴. Finally, proton transfer occurs via the network (W_Zn → W1 → W2 → His64) provided by the protein scaffold.