1 research outputs found
Water Networks in Fast Proton Transfer during Catalysis by Human Carbonic Anhydrase II
Variants of human carbonic anhydrase II (HCA II) with
amino acid
replacements at residues in contact with water molecules in the active-site
cavity have provided insights into the proton transfer rates in this
protein environment. X-ray crystallography and <sup>18</sup>O exchange
measured by membrane inlet mass spectrometry have been used to investigate
structural and catalytic properties of variants of HCA II containing
replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The
rate constants for transfer of a proton from His64 to the zinc-bound
hydroxide during catalysis were 4 and 9 μs<sup>–1</sup> for Y7F and Y7F/N67Q, respectively, compared with a value of 0.8
μs<sup>–1</sup> for wild-type HCA II. These higher values
observed for Y7F and Y7F/N67Q HCA II could not be explained by differences
in the values of the p<i>K</i><sub>a</sub> of the proton
donor (His64) and acceptor (zinc-bound hydroxide) or by the orientation
of the side chain of the proton shuttle residue His64. They appeared
to be associated with a reduced level of branching in the networks
of hydrogen-bonded water molecules between proton shuttle residue
His64 and the zinc-bound solvent molecule as observed in crystal structures
at 1.5–1.6 Å resolution. Moreover, Y7F/N67Q HCA II is
unique among the variants studied in having a direct, hydrogen-bonded
chain of water molecules between the zinc-bound solvent and N<sup>ε</sup> of His64. This study provides the clearest example
to date of the relevance of ordered water structure to rate constants
for proton transfer in catalysis by carbonic anhydrase