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Organization of an efficient carbonic anhydrase: implications for the mechanism based on structure-function studies of a T199P/C206S mutant.
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
Chemistry.
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
Chemistry.
2002 (English)In: Biochemistry, ISSN 0006-2960, Vol. 41, no 24, 7628-35 p.Article in journal (Refereed) Published
Abstract [en]

Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.

Place, publisher, year, edition, pages
2002. Vol. 41, no 24, 7628-35 p.
Keyword [en]
Amino Acid Substitution/*genetics, Bicarbonates/chemistry/metabolism, Binding Sites, Carbon Dioxide/chemistry/metabolism, Carbonic Anhydrases/*chemistry/*genetics/metabolism, Catalysis, Crystallography; X-Ray, Cysteine/genetics, Humans, Mercaptoethanol/chemistry, Mutagenesis; Site-Directed, Proline/genetics, Recombinant Proteins/chemistry/metabolism, Serine/genetics, Structure-Activity Relationship, Substrate Specificity/genetics, Thiocyanates/chemistry/metabolism, Threonine/genetics, Water/chemistry/metabolism
Identifiers
URN: urn:nbn:se:umu:diva-13957PubMedID: 12056894OAI: oai:DiVA.org:umu-13957DiVA: diva2:153628
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2011-01-13Bibliographically approved

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Huang, ShenghuaSauer-Eriksson, Elisabeth
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