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PDBsum entry 2nxr
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References listed in PDB file
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Key reference
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Title
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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.
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Authors
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S.Z.Fisher,
C.Tu,
D.Bhatt,
L.Govindasamy,
M.Agbandje-Mckenna,
R.Mckenna,
D.N.Silverman.
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Ref.
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Biochemistry, 2007,
46,
3803-3813.
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PubMed id
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Abstract
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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.
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