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.
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710.
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Kiefer
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Fierke
D000086:Acetazolamide / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D003545:Cysteine; D006801:Humans; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013912:Threonine; D015032:Zinc / Q000378:metabolism*
D007527:Isoenzymes; D008024:Ligands; D011994:Recombinant Proteins; D013912:Threonine; D003035:Cobalt; D002256:Carbonic Anhydrases; D015032:Zinc; D003545:Cysteine; D000086:Acetazolamide