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.
Kiefer
L L
LL
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710.
Krebs
J F
JF
Paterno
S A
SA
Fierke
C A
CA
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