The role of highly conserved aromatic residues surrounding the zinc binding site of human carbonic anhydrase II (CAII) in determining the metal ion binding specificity of this enzyme has been examined by mutagenesis. Residues F93, F95, and W97 are located along a beta-strand containing two residues that coordinate zinc, H94 and H96, and these aromatic amino acids contribute to the high zinc affinity 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 enhance the copper affinity (up to 100-fold) while decreasing the affinity of both cobalt and zinc, thereby altering the metal binding specificity up to 10(4)-fold. Furthermore, the free energy of the stability of native CAII, determined by solvent-induced denaturation, correlates positively with increased hydrophobicity of the amino acids at positions 93, 95, and 97 as well as with cobalt and zinc affinity. Conversely, increased copper affinity correlates with decreased protein stability. Zinc specificity is therefore enhanced by formation of the native enzyme structure. These data suggest that the hydrophobic cluster in CAII is important for orienting the histidine residues to stabilize metals bound with a distorted tetrahedral geometry and to destabilize the trigonal bipyramidal geometry of bound copper. Knowledge of the structural factors that lead to high metal ion specificity will aid in the design of metal ion biosensors and de novo catalytic sites.

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.

In all metalloenzymes, hydrophobic residues surround the metal binding site. In carbonic anhydrase II (CAII) residues Phe 93 , Phe 95 , and Trp 97 flank two of the three histidines that coordinate zinc to form a hydrophobic cluster beneath the zinc binding site. A library of CAII variants differing in these hydrophobic amino acids was prepared using cassette mutagenesis, then displayed on filamentous phage, and screened for proteins retaining high zinc affinity. Wild-type CAII was enriched 20-fold by selection, and consensus residues at each position were identified from the enriched CAII variants (Ile, Phe, Leu, and Met at position 93; Ile, Leu, and Met at position 95; and Trp and Val at position 97). Highly selected variants have zinc affinity and catalytic activity nearly equal to that of wild-type CAII, indicating that the aromatic residues are not absolutely essential. However, the zinc dissociation rate constant and catalytic activity of the variants correlate with the volume of the amino acids at positions 93, 95, and 97. In summary, metalloenzyme variants displayed on phage can be selected on the basis of metal affinity; such methods will be useful for optimization of metal ion biosensors.Understanding the structural determinants of metal ion affinity in proteins is an important step in the design of protein metal sites. Such sites may be used to stabilize proteins, to regulate the activity of proteins, or for use in biosensors to quantify trace metal ions (see reviews in Refs. 1-3). Because of its high affinity and specificity for zinc, the His 3 metal polyhedron of carbonic anhydrase (CAII) 1 often has been used as a model for designing metal sites in existing proteins (4 -6) and in de novo proteins such as the minibody (7) and four helical bundle protein (8, 9). While the protein metal ligands of CAII have been incorporated into these proteins with coordination geometry very similar to that observed in CAII, these designed metal sites lack the zinc avidity and catalytic activity of biological zinc sites, suggesting that further protein structural factors in CAII contribute to the metal affinity and reactivity of this enzyme.To probe the relationship between protein structure and metal ion affinity and specificity, many of the conserved structural features near the zinc binding site of CAII have been investigated (review see Ref. 10). The refined x-ray crystal structure of CAII (11) 199 (11). This conserved network of residues that form hydrogen bonds with the His ligands enhances zinc affinity (15-17); the Q92A and E117A substitutions both decrease zinc affinity 5-10-fold and increase the zinc dissociation rate constant. Therefore, the protein structure surrounding the zinc binding site of CAII is finely tuned for high zinc affinity and slow rate constants of metal dissociation.A further structural feature observed in all metalloproteins is that the hydrophilic direct metal ligands are embedded within a larger shell of hydrophobic groups (18). In CAII, residues Phe 93 , Phe 95 , and Trp 97 flank the...

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