The important role of active site water in the catalytic mechanism of human carbonic anhydrase II - A semiempirical MO approach to the hydration of CO2

The approach of CO2 to a series of active site model complexes of human car bonic anhydrase II (HCAII) and its catalytic hydration to bicarbonate anion have been investigated using semiempirical MO theory (AM1). The results sh ow that direct nucleophilic attack of zinc-bound hydroxide to the substrate carbon occurs in each model system. Further rearrangement of the bicarbona te complex thus formed via a rotation-like movement of the bicarbonate liga nd can only be found in active site model systems that include at least one additional water molecule. Further refinement of the model complex by addi ng a methanol molecule to mimic Thr-199 makes this process almost activatio nless. The formation of the final bicarbonate complex by an internal (intra molecular) proton transfer is only possible in the simplest of all model sy stems, namely {[Im(3)Zn(OH)](+). CO2}. The energy of activation for this pr ocess, however, is 36.8 kcal.mol(-1) and thus too high for enzymatic cataly sis. Therefore, we conclude that within the limitations of the model system s presented and the level of theory employed, the overall mechanism for the formation of the bicarbonate complex comprises an initial direct nucleophi lic attack of zinc-bound hydroxide to carbon dioxide followed by a rotation -like rearrangement of the bicarbonate ligand via a penta-coordinate Zn2+ t ransition state structure, including the participation of an extra active s ite water molecule.