Metal coordination geometry and mode of action of carbonic anhydrase. Effect of imidazole on the spectral properties of Co(II) and111Cd(II) human carbonic anhydrase B

Abstract

The effect of the inhibitor imidazole on the visible absorption spectrum of Co(II) human carbonic anhydrase B and on the perturbed angular correlation spectrum of111Cd(II) human carbonic anhydrase B have been studied at various pH values. It is demonstrated that imidazole binds both to the111Cd(II) and Co(II) enzymes and perturbs their respective spectra. Furthermore, spectral titrations of Co(II) with imidazole reveals binding constants similar to those determined kinetically for both the Zn(II) and the Cd(II) enzymes. Imidazole binds to both the low and high pH forms of the enzyme, and have only a weak effect on the activity linked ionization. The perturbed angular correlation results suggest that the human carbonic anhydrase B imidazole complex is five coordinated with a hydrolytic water molecule, imidazole and from the protein part His-94, His-96 and His-119 as the ligands. The effect of acetazolamide binding of the perturbed angular correlation spectrum suggest, that this inhibitor also forms a five coordinated complex in which the NH− and an oxygen atom of the sulfonamide group displace one or two water molecules. Detailed analysis of the perturbed angular correlation data for111Cd(II) human carbonic anhydrase B suggests that the low pH form of the enzyme is four coordinated with a water molecule as the fourth ligand, whereas the high pH and active form of the enzyme might be five coordinated with a hydroxyl ion and a water molecule as the fourth and fifth ligands, respectively. A mechanism for CO2 hydration is proposed in which the metal is five coordinated and functions both as aLewis acid, activating CO2 through direct coordination, and by activation of a coordinated water molecule for nucleophilic attack on CO2.