title: Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II.
abstract: The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹⁸O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had no effect on the interconversion of bicarbonate and CO₂, but in some cases caused enhancements in the rate constant of proton transfer by nearly 10-fold. The variant Y7I HCA II enhanced intramolecular proton transfer approximately twofold; its structure was determined by X-ray crystallography at 1.5 Å resolution. No changes were observed in the ordered solvent structure in the active-site cavity or in the conformation of the side chain of the proton shuttle His64. However, the first 11 residues of the amino-terminal chain in Y7I HCA II assumed an alternate conformation compared with the wild type. Differential scanning calorimetry showed variants at position 7 had a melting temperature approximately 8 °C lower than that of the wild type.
authors.affiliation: Department of Pharmacology, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Rose
authors.initials: R
authors.lastname: Mikulski
authors.forename: Balendu Sankara
authors.initials: BS
authors.lastname: Avvaru
authors.forename: Chingkuang
authors.initials: C
authors.lastname: Tu
authors.forename: Nicolette
authors.initials: N
authors.lastname: Case
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
authors.forename: David N
authors.initials: DN
authors.lastname: Silverman
mesh_terms: D019943:Amino Acid Substitution; D001639:Bicarbonates / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002245:Carbon Dioxide / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001639:Bicarbonates; D011522:Protons; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D014443:Tyrosine; D024402:Carbonic Anhydrase II