Examination of the role of Gln-158 in the mechanism of CO2 hydration catalyzed by β-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a variant of Arabidopsis thaliana β-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human α-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO2–HCO3− interconversion in α-carbonic anhydrase but this hypothesis is not directly testable in that enzyme. Kinetic studies of a variant of the functionally equivalent residue in A. thaliana β-carbonic anhydrase provide direct evidence for the role of this residue in β-carbonic anhydrase. Namely, the mutation of Gln-158 to Ala results in a significant decrease in the maximal kcat/Km (33% of wild type) at steady state and the maximal rate of CO2–HCO3− exchange at chemical equilibrium as measured by R1/[E] (7% of wild type), while leaving the maximal rate of H+ transfer, as measured by kcat at steady state, or RH2O/[E] at chemical equilibrium, largely unaffected.