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

• Published in: Chemistry : a European journal Vol. 30; no. 24; pp. e202304367 - n/a
• Main Authors: Borghesani, Valentina, Zastrow, Melissa L., Tolbert, Audrey E., Deb, Aniruddha, Penner‐Hahn, James E., Pecoraro, Vincent L.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 25-04-2024
• Subjects: Carbonic anhydrase; Carbonic anhydrase II; Carbonic anhydrases; Carbonic Anhydrases - chemistry; Carbonic Anhydrases - metabolism; Catalysis; Catalytic Domain; Cobalt; Cobalt - chemistry; Coordination; Coordination Complexes - chemistry; Coordination Complexes - metabolism; Enzyme activity; Esterases - chemistry; Esterases - metabolism; Histidine; Humans; Hydrogen-Ion Concentration; Hydrolysis; Imidazole; Kinetics; Metals; Nitrophenols - chemistry; Nitrophenols - metabolism; Peptides and proteins; pH effects; Protein kinase A; Spectroscopy; Substitution reactions; Zinc; Zinc - chemistry; Hydrolysis; Peptides and proteins; Enzyme activity; Coordination; Metals
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.202304367

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)3n+ 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 pKa for the conversion of 10.3. We assign this conversion to the formation of a 5‐coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), 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.
The Co(II) analogue of the designed peptide Zn(II)(TRIL23H)3 is made as a spectroscopic probe for the carbonic anhydrase activity exhibited by this de novo designed system. The coordination geometry of Co(II) and Zn(II) are vastly different at high and low pH. Nonetheless, the Co(II) analogue at high pH exceeds the catalytic efficiency of the parent Zn(II) protein.