Engineering a cysteine ligand into the zinc binding site of human carbonic anhydrase II.

Substitution of cysteine for threonine-199, the amino acid which hydrogen bonds with zinc-bound hydroxide in wild-type carbonic anhydrase II (CAII), leads to the formation of a new His3Cys zinc coordination polyhedron. The optical absorption spectrum of the Co(2+)-substituted threonine-199-->cysteine (T199C) variant and the three-dimensional structure [Ippolito, J. A., & Christianson, D. W. (1993) Biochemistry (following paper in this issue)] indicate that the new thiolate side chain coordinates to the metal ion, displacing the metal-bound solvent molecule. The engineered thiolate ligand increases zinc binding (4-fold) and decreases catalytic activity substantially (approximately 10(3)-fold) but not completely. However, this residual activity is due to an active species containing a zinc-bound solvent ligand with the cysteine-199 side chain occupying an alternate conformation. The equilibrium between these conformers reflects the energetic balance between the formation of the zinc-thiolate bond and structural rearrangements in the Ser-197-->Cys-206 loop necessary to achieve this metal coordination. This designed His3Cys metal polyhedron may mimic the zinc binding site in the matrix metalloproteinase prostromelysin.