title: Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II.
abstract: Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH-) and the proton-shuttling residue His64. This distance (approximately 7.5 A) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (approximately 1.0 A) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 A resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble, France.
authors.forename: Monika
authors.initials: M
authors.lastname: Budayova-Spano
authors.forename: S Zoë
authors.initials: SZ
authors.lastname: Fisher
authors.forename: Marie Thérèse
authors.initials: MT
authors.lastname: Dauvergne
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.forename: David N
authors.initials: DN
authors.lastname: Silverman
authors.forename: Dean A A
authors.initials: DA
authors.lastname: Myles
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
mesh_terms: D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D024402:Carbonic Anhydrase II / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D003903:Deuterium; D006801:Humans; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D003903:Deuterium; D024402:Carbonic Anhydrase II