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Organization of an efficient carbonic anhydrase: Implications for the mechanism based on structure-function studies of a T199P/C206S mutant
Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
2002 (English)In: Biochemistry, ISSN 0006-2960, Vol. 41, no 24, 7628-7635 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 ß-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 ß-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, 7628-7635 p.
National Category
Engineering and Technology
URN: urn:nbn:se:liu:diva-46971DOI: 10.1021/bi020053oOAI: diva2:267867
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2011-01-13

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Jonsson, Bengt-Harald
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