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

Biochemistry. 2000 Nov 14;39(45):13687-94. doi: 10.1021/bi001649j.


Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) 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, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities 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 conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Amino Acids / chemistry*
  • Binding Sites
  • Carbonic Anhydrases / chemistry*
  • Carbonic Anhydrases / metabolism*
  • Cobalt / metabolism
  • Copper / metabolism
  • Crystallography, X-Ray
  • Humans
  • Leucine / chemistry
  • Metals, Heavy / metabolism*
  • Methionine / chemistry
  • Phenylalanine / chemistry
  • Serine / chemistry
  • Structure-Activity Relationship
  • Substrate Specificity
  • Tryptophan / chemistry
  • Valine / chemistry
  • Zinc / metabolism


  • Amino Acids
  • Metals, Heavy
  • Cobalt
  • Serine
  • Phenylalanine
  • Copper
  • Tryptophan
  • Methionine
  • Carbonic Anhydrases
  • Leucine
  • Valine
  • Zinc

Associated data

  • PDB/1FQL
  • PDB/1FQM
  • PDB/1FQN
  • PDB/1FQR
  • PDB/1FR4
  • PDB/1FR7
  • PDB/1FSN
  • PDB/1FSQ
  • PDB/1FSR