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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.

The crystal structure of a human carbonic anhydrase II (CAII) variant, cis-proline-202-->alanine (P202A), has been determined at 1.7-A resolution, indicating that the wild-type geometry, including the cis-peptidyl linkage, is retained upon substitution of proline by alanine. The CO2 hydrase activity and affinity for sulfonamide inhibitors of P202A CAII are virtually identical to those of wild type. However, the substitution of cis-alanine for cis-proline decreases the stability of the folded state by approximately 5 kcal mol-1 relative to both the unfolded state and an equilibrium intermediate in guanidine hydrochloride-induced denaturation. This destabilization can be attributed mainly to the less favorable cis/trans equilibrium of Xaa-alanine bonds compared to Xaa-proline bonds in the denatured state although other factors, including increased conformational entropy of the denatured state and decreased packing interactions in the native state, also contribute to the observed destabilization. The high catalytic activity of P202A CAII illustrates that unfavorable local conformations are nonetheless endured to satisfy the precise structural requirements of catalysis and ligand binding in the CAII active site.

The Design of Green Oxidants -[39 refs.]. -(COLLINS, TER-RENCE J.; GORDON-WYLIE, SCOTT W.; BARTOS, MICHAEL J.; HORWITZ, COLIN P.; WOOMER, CHRISTINE G.; WILLIAMS, STACY A.; PATTERSON, ROBERT E.; VUOCOLO, LEONARD D.; PATERNO, STEVEN A.; STRAZISAR, STEPHANIE A.; ET AL.; Green Chem. (1998) 46-71; Dep. Chem., Carnegie Mellon Univ., Pittsburgh, PA 15213, USA; EN)