Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin-lattice relaxation studies

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Abstract
N.tau.-carboxymethylhistidine-200 human carbonic anhydrase B [EC 4.2.1.1] was previously prepared using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. Chemical shift studies of the Zn-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the Zn enzyme at 3 frequencies (15.04, 25.15 and 90.5 MHz) are reported here. The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the Zn. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed Zn-carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data was interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The catalytic consequences of the carboxymethylation may be due to the proposed Zn-carboxylate coordination and need not result from the disruption of any role that his-200 might play in the catalytic mechanism.

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