title: Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II.
abstract: 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.
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Cox
authors.forename: J A
authors.initials: JA
authors.lastname: Hunt
authors.forename: K M
authors.initials: KM
authors.lastname: Compher
authors.forename: C A
authors.initials: CA
authors.lastname: Fierke
authors.forename: D W
authors.initials: DW
authors.lastname: Christianson
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D018360:Crystallography, X-Ray; D006801:Humans; D007930:Leucine / Q000737:chemistry; D019216:Metals, Heavy / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry; D014633:Valine / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
chemical_list: D000596:Amino Acids; D019216:Metals, Heavy; D003035:Cobalt; D012694:Serine; D010649:Phenylalanine; D003300:Copper; D014364:Tryptophan; D008715:Methionine; D002256:Carbonic Anhydrases; D007930:Leucine; D014633:Valine; D015032:Zinc