Histidine 64 is not required for high CO2 hydration activity of human carbonic anhydrase II.
To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a 'proton shuttle group' during catalysis of CO2-HCO3- interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site-directed mutagenesis. All these variants turned out to have high CO2 hydration activities. The kcat values at pH 8.8 and 25 degrees 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 pKa 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 pKa shifted to 8.4.
D001665:Binding Sites; D002245:Carbon Dioxide / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005333:Fetus; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011485:Protein Binding
D002245:Carbon Dioxide; D006639:Histidine; D004247:DNA; D002256:Carbonic Anhydrases