Marked zinc activation of ester hydrolysis by a mutation, 67-His (CAT) to Arg (CGT), in the active site of human carbonic anhydrase I

Eight carbonic anhydrase (CA) or CA-like genes are now known to be expressed in humans. Although the principle catalytic role of the carbonic anhydrases is the reversible hydration of CO sub(2), the CA I and CA II isozymes also possess esterase activity toward p-nitrophenyl acetate, alpha - and beta -naphthyl acetate, and 2-hydroxy-5-nitro- alpha -toluene sulfonic acid sultone. These findings suggested that free zinc ions dissociating from the Blue RR complex were activating the esterase activity of the mutant CA I molecule. The esterase activity toward beta -naphthyl acetate was shown to be increased 10-fold at a Zn super(2+) concentration of 1 x 10 super(-4) M. At this concentration of Zn super(2+), the HCO sub(3) super(-) dehydration activities of the mutant and normal CA I were decreased by 40% and 20%, respectively. Further studies indicated that the activation was Zn super(2+) specific because a similar activation was not observed in the presence of other metal divalent cations, i.e., Co super(2+), Cu super(2+), Mg super(2+), and Ni super(2+). Furthermore, it appeared to be specific for naphthyl acetates and not for other ester substrates of CA I, i.e., p-nitrophenyl acetate, alpha -hydroxy-5-nitro- alpha -toluene sulfonic acid sultone. We report an amino acid substitution at the active site of CA I Michigan-1 that appears to be responsible for the novel modification of one of the esterase activities of this mutant enzyme. This is a rare example of true metal ion activation resulting from a point mutation.