Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site-specific mutagenesis

Rates of hydrolysis of 4-, 3-, and 2-nitrophenyl acetate and 4-nitrophenyl propionate catalyzed by wild-type and mutant forms of human carbonic anhydr ase II have been measured. The results show that the mutations Tyr7 --> Phe and Ala65 --> Leu lead to activity enhancements with all the investigated substrates, but then is no significant effect on the specificity. In contra st, some mutations at sequence position 200 have large effects on specifici ty. For example, while the mutation Thr200 --> Gly results in a threefold i ncrease of the rate of hydrolysis of 4-nitrophenyl acetate, the activity is enhanced 10 times with the meta-substituted substrate and 380 times with t he ortho-substituted substrate. These results are interpreted in terms of t he removal in the mutant of a steric interference between the 2-NO2 group, in particular, and the side chain of Thr200. Mutants involving residues lin ing a hydrophobic pocket near the catalytically essential zinc ion have als o been investigated. The most pronounced effect on specificity was found fo r the Val143 --> Gly mutant. This mutation leads to a sixfold decrease of t he rate of hydrolysis of 4-nitrophenyl acetate but a 20-fold increase of th e activity with the propionyl ester as substrate. These results suggest tha t the side chain of Val143 interferes sterically with the acyl moiety of 4- nitrophenyl propionate. Based on these results, we have constructed a hypot hetical model of the location of these ester substrates in the enzymic acti ve site.