Human carbonic anhydrase II (hCAII) is a zinc metalloenzyme that is abundant in erythrocytes (Lindskog, 1997
). The enzymatic reaction, the hydration of carbon dioxide to bicarbonate or the reverse dehydration of bicarbonate, occurs at the zinc ion.
The enzyme is roughly spherical in shape and the active site lies within a conical cleft about 15 Å deep; the zinc ion lies at the bottom of this cleft and is tetrahedrally liganded by His94, His96, His119 and a hydroxide ion at physiological pH (Håkansson et al.
; Stams & Christianson, 2000
). The zinc-bound hydroxide forms a hydrogen bond to the hydroxyl group of Thr199, which in turn forms a hydrogen bond to Glu106. This hydrogen-bond network, along with the so-called ‘deep water’ molecule located at the mouth of the substrate-binding pocket, orients the zinc-bound nucleophilic hydroxide for optimal attack on the substrate. A second hydrogen-bond network engages the zinc-bound hydroxide and the imidazole side chain of His64 through two intervening solvent molecules and this network is likewise important for catalysis. His64 acts as a proton shuttle in catalysis, in which it accepts the proton product (via
the bridging solvent molecules) from zinc-bound water as the zinc-bound hydroxide is regenerated; subsequently, the proton product is passed along to buffers. Conformational mobility has been noted for the imidazole ring of the His64 side chain. It has been noted in either an ‘in’ (facing towards zinc) or an ‘out’ (facing away from zinc) position. This facilitates proton transfer between the active-site waters and solvent water at the mouth of the active-site cavity (Tripp et al.
Inhibitors such as inorganic anions and aromatic/heterocyclic sulfonamides bind within the carbonic anhydrase (CA) active site by coordinating to the metal ion either in a tetrahedral geometry, replacing the zinc-bound water/hydroxide, or in a pentacoordinated mode, adding to the coordination sphere without displacing the zinc-bound water molecule (Pastorekova et al.
; Supuran & Scozzafava, 2007
). The crystal structures of the acetate complexes of wild-type hCAII (PDB code 1cay
) and E106Q mutant hCAII (PDB code 1caz
) have previously been solved to 2.1 and 1.9 Å resolution, respectively (Håkansson et al.
). Both crystallized in a typical manner in space group P
, with unit-cell parameters a
= 42.7, b
= 41.7, c
= 73.0 Å, β = 104.6°. The altered active-site hydrogen-bond network caused by the mutation resulted in a different binding mode of the inhibitor in the two complexes.