2NWO: Structural and Kinetic Effect of Hydrophobic Mutations in the Active Site of Human Carbonic Anhydrase II

Catalysis of the hydration of CO2 by human carbonic anhydrase isozyme II (HCA II) is sustained at a maximal catalytic turnover of 1 mus-1 by proton transfer between a zinc-bound solvent and bulk solution. This mechanism of proton transfer is facilitated via the side chain of His64, which is located 7.5 A from the zinc, and mediated via intervening water molecules in the active-site cavity. Three hydrophilic residues that have previously been shown to contribute to the stabilization of these intervening waters were replaced with hydrophobic residues (Y7F, N62L, and N67L) to determine their effects on proton transfer. The structures of all three mutants were determined by X-ray crystallography, with crystals equilibrated from pH 6.0 to 10.0. A range of changes were observed in the ordered solvent and the conformation of the side chain of His64. Correlating these structural variants with kinetic studies suggests that the very efficient proton transfer (approximately 7 micros-1) observed for Y7F HCA II in the dehydration direction, compared with the wild type and other mutants of this study, is due to a combination of three features. First, in this mutant, the side chain of His64 showed an appreciable inward orientation pointing toward the active-site zinc. Second, in the structure of Y7F HCA II, there is an unbranched chain of hydrogen-bonded waters linking the proton donor His64 and acceptor zinc-bound hydroxide. Finally, the difference in pKa of the donor and acceptor appears favorable for proton transfer. The data suggest roles for residues 7, 62, and 67 in fine-tuning the properties of His64 for optimal proton transfer in catalysis.
PDB ID: 2NWODownload
MMDB ID: 45683
PDB Deposition Date: 2006/11/16
Updated in MMDB: 2007/10
Experimental Method:
x-ray diffraction
Resolution: 1.7  Å
Source Organism:
Similar Structures:
Biological Unit for 2NWO: monomeric; determined by author
Molecular Components in 2NWO
Label Count Molecule
Protein (1 molecule)
Carbonic Anhydrase 2(Gene symbol: CA2)
Molecule annotation
Chemical (1 molecule)
* Click molecule labels to explore molecular sequence information.

Citing MMDB