A carbon-13 NMR comparative study of metal ion substitutions in human carbonic anhydrase I carboxymethylated at active-site Histidine-200

Human carbonic anhydrase I (EC 4.2.1.1), the low-activity isozyme, has a reactive active-site Histidine-200 that is known to be specifically modified at N τ with haloacetates. Using [1- 13C]bromoacetate, we previously introduced a highly sensitive 13C NMR probe into the active site of the enzyme and studied the interaction of the carboxymethyl carboxylate with the active-site zinc, as well as the ionization properties of the carboxymethylated histidine-200 side chain. In the present work, these studies have been extended to metalloderivatives of the enzyme in which the intrinsic zinc has been replaced by Cd 2+, Hg 2+, and Co 2+. In the former two metals, spin- 1 2 isotopes ( 113Cd and 199Hg) in the absence of inhibitory halides were utilized to search for two-bond spin-spin couplings in the spectrum of the 13C-enriched carboxymethyl carboxylate under conditions where coordination exists in the Zn and Co derivatives. The absence of splittings and titration studies of the chemical shift of the resonance both established the absence of coordination. The pH dependence of the carboxylate, which reflects the ionization of the CmHis-200 ring, was observed in the presence and absence of bound inhibitors. Marked differences were seen among the four metalloderivatives in all these properties, suggesting great sensitivity of the active site to the nature of the metal inserted. The data suggest extreme caution in extrapolating results from metal ion substitution studies to the native zinc enzyme, and may reflect functional significance of this sensitivity in the catalysis.