Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II.
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Budayova-Spano M, Fisher SZ, Dauvergne MT, Agbandje-McKenna M, Silverman DN, Myles DA, McKenna R
Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Jan 1;62(Pt 1):6-9. Epub 2005 Dec 16.
- PubMed ID
- 16511248 [ View in PubMed]
- 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.