2nxt
Structural and kinetic effects of hydrophobic mutations in the active site of human carbonic anhydrase II
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OverviewOverview
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.
About this StructureAbout this Structure
2NXT is a Single protein structure of sequence from Homo sapiens with as ligand. Active as Carbonate dehydratase, with EC number 4.2.1.1 Full crystallographic information is available from OCA.
ReferenceReference
Speeding up proton transfer in a fast enzyme: kinetic and crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase II., Fisher SZ, Tu C, Bhatt D, Govindasamy L, Agbandje-McKenna M, McKenna R, Silverman DN, Biochemistry. 2007 Mar 27;46(12):3803-13. Epub 2007 Mar 2. PMID:17330962
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