Co(II) Substitution Enhances the Esterase Activity of a de Novo Designed Zn(II) Carbonic Anhydrase

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d⁷ electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H₂O/OH^-)]_N(TRIL2WL23H)₃ ⁿ⁺ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH^-)]_N(TRIL2WL23H)₃ ⁺ peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pK_a for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(N_imid)₃(OH)(H₂O) species. The pH dependent kinetic analysis indicates the maximal rate (k_cat), and thus the catalytic efficiency (k_cat/K_m), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.