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Organization of an Efficient Carbonic Anhydrase: Implications for the Mechanism Based on Structure−Function Studies of a T199P/C206S Mutant
- Source :
- Biochemistry. 41:7628-7635
- Publication Year :
- 2002
- Publisher :
- American Chemical Society (ACS), 2002.
-
Abstract
- 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.
- Subjects :
- Threonine
Proline
Stereochemistry
Carbonic anhydrase II
Mutant
Mechanism based
Crystallography, X-Ray
Biochemistry
Catalysis
Substrate Specificity
Structure-Activity Relationship
Carbonic anhydrase
Serine
Humans
Cysteine
Catalytic efficiency
Carbonic Anhydrases
Mercaptoethanol
Binding Sites
biology
Chemistry
Structure function
Water
Active site
Carbon Dioxide
Lyase
Recombinant Proteins
Bicarbonates
Amino Acid Substitution
Mutagenesis, Site-Directed
biology.protein
Thiocyanates
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 41
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....b2e7c1a198f2439f8713fa1c001064cf