Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (C AII) 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, H 94 and H96. The aromatic amino acids contribute to the high zinc affinity a nd slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C . A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromati c amino acids with smaller side chains enhances Cu2+ affinity while decreas ing Co2+ and Zn2+ affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (19 99) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bou nd F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new c avities 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 confor mational change of direct metal ligand H94 as well as indirect (i.e., "seco nd shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine l igands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound va riant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geomet ry; F93S/F95L/W97M CAII binds Co2+ with trigonal bipyramidal coordination g eometry due to the addition of azide anion to the metal coordination polyhe dron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second so lvent molecule to the metal coordination polyhedron. The key finding of thi s work is that aromatic core residues serve as anchors that help to preorie nt direct and second-shell ligands to optimize zinc binding geometry and de stabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compa red to small molecule chelators.