Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II.
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.
Budayova-Spano
Monika
M
European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble, France.
Fisher
S Zoë
SZ
Dauvergne
Marie Thérèse
MT
Agbandje-McKenna
Mavis
M
Silverman
David N
DN
Myles
Dean A A
DA
McKenna
Robert
R
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
D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D003903:Deuterium; D024402:Carbonic Anhydrase II