An approach to water molecule dynamics associated with motion of catalytic moiety

A water bridge composed of several water molecules between the catalytic moieties, His64 and the zinc-bound solvent, in human carbonic anhydrase II (hCAII) is disrupted when the inhibitor acetazolamide (ACZ) binds to the zinc ion, according to the crystallographic structure of the ACZ-hCAII complex. In this structure, the ACZ methyl group is far (∼10 Å) from the His64. However, this binding causes an 1H NMR chemical shift change (∼1 ppm) in His64 in solution. This suggests two alternative mechanisms: a) the ACZ methyl group may be closer to His64 in the complex in solution, compared to the crystal, or b) the disruption of the water bridge might cause the His64 to move or behave in a different manner. The binding of ACZ to the enzyme in solution was examined by observing the NMR signals of the 13C-labeled ACZ methyl group in the ACZ-hCAII complex. The 13C signals of the free and bound forms were detected. In the bound form, the signal for the acetamide group was pH dependent, whereas the sulfonamide group signal was pH independent. Some 13C-filtered NOE signals were observed, although none of the signals were related to the His64 chemical shift. Based on these observations, we suggest that the position or motion of His64 is associated with disruption of the water bridge in the ACZ-hCAII complex in solution, which could change the 1H chemical shift.