| Abstract
| - The role of highly conserved aromatic residues surrounding the zinc binding site of humancarbonic anhydrase II (CAII) in determining the metal ion binding specificity of this enzyme has beenexamined by mutagenesis. Residues F93, F95, and W97 are located along a β-strand containing tworesidues that coordinate zinc, H94 and H96, and these aromatic amino acids contribute to the high zincaffinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol.Chem.272, 20364−20372]. Substitutions of these aromatic amino acids with smaller side chains enhancethe copper affinity (up to 100-fold) while decreasing the affinity of both cobalt and zinc, thereby alteringthe metal binding specificity up to 104-fold. Furthermore, the free energy of the stability of native CAII,determined by solvent-induced denaturation, correlates positively with increased hydrophobicity of theamino acids at positions 93, 95, and 97 as well as with cobalt and zinc affinity. Conversely, increasedcopper affinity correlates with decreased protein stability. Zinc specificity is therefore enhanced by formationof the native enzyme structure. These data suggest that the hydrophobic cluster in CAII is important fororienting the histidine residues to stabilize metals bound with a distorted tetrahedral geometry and todestabilize the trigonal bipyramidal geometry of bound copper. Knowledge of the structural factors thatlead to high metal ion specificity will aid in the design of metal ion biosensors and de novo catalyticsites.
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