Kinetic characterization of wild-type and proton transfer-impaired variants of β-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a truncated, recombinant form of β-carbonic anhydrase from Arabidopsis thaliana. The wild-type enzyme and two site-directed variants, H216N and Y212F, have been kinetically characterized both at steady state by stopped-flow spectrophotometry and at chemical equilibrium by 18O isotope exchange methods. The wild-type enzyme has a maximal kcat for CO2 hydration of 320 ms−1 and is rate limited by proton transfer involving two residues with apparent pKa values of 6.0 and 8.7. The mutant enzyme H216N has a maximal kcat at high pH that is 43% that of wild type, but is only 5% that of wild type at pH 7.0. 18O exchange studies reveal that the effect of the mutations H216N or Y212F is primarily on proton transfer steps in the catalytic mechanism and not in the rate of CO2–HCO3− exchange. These results suggest that residues His-216 and Tyr-212 are both important for efficient proton transfer in A. thaliana carbonic anhydrase.