Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II
- European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France)
- Department of Biochemistry and Molecular Biology, PO Box 100245, University of Florida, Gainesville, FL 32610 (United States)
- Department of Pharmacology and Therapeutics, PO Box 100267, University of Florida, Gainesville, FL 32610 (United States)
This article reports the production, crystallization and X-ray structure determination of perdeuterated human carbonic anhydrase (HCA II). The refined structure is shown to be highly isomorphous with hydrogenated HCA II, especially with regard to the active site architecture and solvent network. 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 (H{sub 2}O/OH{sup −}) and the proton-shuttling residue His64. This distance (∼7.5 Å) 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 (∼1.0 Å) 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 Å 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.
- OSTI ID:
- 22356243
- Journal Information:
- Acta Crystallographica. Section F, Vol. 62, Issue Pt 1; Other Information: PMCID: PMC2150928; PMID: 16511248; PUBLISHER-ID: fw5056; OAI: oai:pubmedcentral.nih.gov:2150928; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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