title: Organization of an efficient carbonic anhydrase: implications for the mechanism based on structure-function studies of a T199P/C206S mutant.
abstract: Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.
authors.affiliation: Umeå Centre for Molecular Pathogenesis, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Shenghua
authors.initials: S
authors.lastname: Huang
authors.forename: Björn
authors.initials: B
authors.lastname: Sjöblom
authors.forename: A Elisabeth
authors.initials: AE
authors.lastname: Sauer-Eriksson
authors.forename: Bengt-Harald
authors.initials: BH
authors.lastname: Jonsson
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D001639:Bicarbonates / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D006801:Humans; D008623:Mercaptoethanol / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013861:Thiocyanates / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000235:genetics; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D001639:Bicarbonates; D011994:Recombinant Proteins; D013861:Thiocyanates; D014867:Water; D002245:Carbon Dioxide; D013912:Threonine; D012694:Serine; D008623:Mercaptoethanol; D011392:Proline; D002256:Carbonic Anhydrases; D003545:Cysteine; C031760:thiocyanate