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Histidine 64 is not required for high CO 2 hydration activity of human carbonic anhydrase II
Author(s) -
Forsman Cecilia,
Behravan Gity,
Jonsson Bengt-Harald,
Liang Zhi-wei,
Lindskog Sven,
Ren Xilin,
Sandström Jan,
Wallgren Katarina
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)81156-6
Subject(s) - chemistry , histidine , carbonic anhydrase , enzyme kinetics , alanine , enzyme , stereochemistry , catalysis , carbonic anhydrase ii , active site , lysine , intramolecular force , glutamine , hydrolysis , glutamic acid , amino acid , medicinal chemistry , biochemistry
To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a ‘proton shuttle group’ during catalysis of CO 2 ‐HCO − 3 interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site‐directed mutagenesis. All these variants turned out to have high CO 2 hydration activities. The k cat values at pH 8.8 and 25°C were only reduced by 1.5–3.5‐fold compared to the unmodified enzyme. These results show that intramolecular proton transfer via His 64 is not a dominating pathway in the catalytic reaction. The variants also catalyze the hydrolysis of 4‐nitrophenyl acetate. The p K a values for the activity‐controlling group are between 6.8 and 7.0 for all studied forms of the enzyme except the Glu 64 variant which shows a complex pH dependence with the major p K a shifted to 8.4