Importance of the conserved active-site residues Tyr7, Glu106 and Thr199 for the catalytic function of human carbonic anhydrase II.

The catalytic mechanism of carbonic anhydrase includes the reaction of a zinc-bound hydroxide ion with the CO2 substrate. This hydroxide ion is part of a hydrogen-bonded network involving the conserved amino acid residues Thr199, Glu106 and Tyr7. To investigate the functional importance of these residues, a number of site-specific mutants have been made. Thus, Thr199 has been changed to Ala, Glu106 to Ala, Gln and Asp, and Tyr7 to Phe. The effects of these mutations on catalyzed CO2 hydration and ester hydrolysis have been measured, as well as the binding of some inhibitors. The results show that the CO2 hydration activity of the mutant with Phe7 is only marginally reduced, whereas the esterase activity is larger than that of unmodified enzyme. It is concluded that Tyr7 is not a functionally required element of the hydrogen-bonded network. The CO2 hydration activity (kcat as well as kcat/Km) and the esterase activity of the mutant with Ala199 are reduced about 100-fold. The affinity for the sulfonamide inhibitor, dansylamide, is only slightly reduced while the mutant has an enhanced affinity for bicarbonate and the anionic inhibitor, SCN-. The activities of the mutants with Ala106 and Gln106 are also reduced. The reduction of the esterase activity is about 100-fold, while kcat for CO2 hydration has decreased by a factor of 1000. The parameter kcat/Km is only about one order of magnitude smaller for these mutants than for the unmodified enzyme. The binding of dansylamide and another sulfonamide inhibitor, acetazolamide, are about 20-times weaker to the mutant with Gln106 than to unmodified enzyme. These results suggest important roles for Thr199 and Glu106 in carbonic anhydrase catalysis. The mutant with Asp106 is almost fully active suggesting that the structure has undergone a compensatory change to maintain the interaction between residue 106 and Thr199.