Molecular determinants of S-glutathionylation of carbonic anhydrase 3.

Carbonic anhydrase 3 is easily S-glutathionylated in vivo and in vitro. The protein has two surface-exposed cysteine residues that can be modified. We found that Cys186 is more readily glutathionylated than Cys181. We studied a series of site-specific mutants to identify the residues that interact with Cys186 to make its thiol more reactive. We found that Lys211 is responsible for lowering the pKa of Cys186. We also found that two acidic residues, Asp188 and Glu212, interact with the thiol and actually decrease its reactivity. We speculate that conformational changes that alter the interaction with these three residues provide a mechanistic basis for modulation of the susceptibility of carbonic anhydrase 3 to glutathionylation.