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Organization of an efficient carbonic anhydrase: implications for the mechanism based on structure-function studies
Shenghua Huang1, Björn Sjöblom, A Elisabeth Sauer-Eriksson
1Umeå Centre for Molecular Pathogenesis, Umeå University, SE-901 87 Umeå, Sweden.
Insights
Substituting proline for threonine in human carbonic anhydrase II (HCA II) drastically reduces catalytic efficiency. Structural analysis reveals novel ligand binding in the mutant, impacting enzyme mechanism and evolution.
Area of Science:
- Biochemistry
- Structural Biology
- Enzyme Kinetics
Background:
- Human carbonic anhydrase II (HCA II) is a crucial enzyme in physiological processes.
- The active site residue Threonine 199 (Thr199) plays a key role in HCA II's catalytic activity.
- Mutating Thr199 to Proline (Pro) significantly impairs enzyme function.
Purpose of the Study:
- To investigate the structural and mechanistic consequences of the Thr199Pro substitution in HCA II.
- To elucidate the binding interactions of substrates and inhibitors with the T199P/C206S HCA II variant.
- To understand the evolutionary role of Thr199 in substrate orientation.
Main Methods:
- X-ray crystallography was employed to determine the structures of the T199P/C206S HCA II variant.
- Structures were solved in complex with bicarbonate, thiocyanate, and beta-mercaptoethanol.
- Ligand binding modes and active site environments were analyzed.
Main Results:
- The T199P/C206S mutation reduced HCA II catalytic efficiency by approximately 3000-fold.
- Novel binding interactions were observed for bicarbonate, thiocyanate, and beta-mercaptoethanol in the mutant active site.
- Beta-mercaptoethanol, typically not an inhibitor, bound tetrahedrally to the zinc ion.
- Thiocyanate exhibited tetrahedral coordination to the zinc ion, differing from wild-type pentacoordinated binding.
- Bicarbonate bound in a more hydrophilic region, utilizing different oxygen coordination sites compared to wild-type HCA II.
Conclusions:
- The Thr199Pro substitution dramatically alters the HCA II active site architecture and ligand binding properties.
- The observed novel binding modes provide insights into the enzyme's catalytic mechanism and substrate recognition.
- These findings highlight the critical role of Thr199 in the evolutionary adaptation of HCA II for efficient substrate binding and catalysis.
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.