Histidine 64 is not required for high CO 2 hydration activity of human carbonic anhydrase II

Abstract

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