Introduction of Histidine Analogs Leads to Enhanced Proton Transfer in Carbonic Anhydrase V

• Published in: Archives of biochemistry and biophysics 1999-01, Vol.361 (2), p.264-270
• Main Authors: Earnhardt, J.Nicole, Wright, S.Kirk, Qian, Minzhang, Tu, Chingkuang, Laipis, Philip J., Viola, Ronald E., Silverman, David N.
• Format: Article
• Language: English
• Subjects: Animals; carbon dioxide; carbonic anhydrase; Carbonic Anhydrases - genetics; Carbonic Anhydrases - metabolism; Catalysis; chemical modification; Cysteine - genetics; Cysteine - metabolism; Histidine - analogs & derivatives; Histidine - metabolism; Mice; Mice, Inbred BALB C; Mutagenesis, Site-Directed; Oxygen Isotopes; proton transfer; Protons; Substrate Specificity; unnatural amino acid
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1998.0984

The rate-limiting step in the catalysis of the hydration of CO2by carbonic anhydrase involves transfer of protons between zinc-bound water and solution. This proton transfer can be enhanced by proton shuttle residues within the active-site cavity of the enzyme. We have used chemical modulation to provide novel internal proton transfer groups that enhance catalysis by murine carbonic anhydrase V (mCA V). This approach involves the site-directed mutation of a targeted residue to a cysteine which is then subsequently reacted with an imidazole analog containing an appropriately positioned leaving group. Compounds examined include 4-bromoethylimidazole (4-BEI), 2-chloromethylimidazole (2-CMI), 4-chloromethylimidazole (4-CMI), and a triazole analog. Two sites in mCA V, Lys 91 and Tyr 131, located on the rim of the active-site cavity have been targeted for the introduction of these imidazole analogs. Modification of the introduced Cys 131 with 4-BEI and 4-CMI resulted in enhancements of up to threefold in catalytic activity. The pH profiles indicate the presence of a new proton shuttle residue of pKanear 5.8, consistent with the introduction of a functional proton transfer group into the active site. This is the first example of incorporation by chemical modification of an unnatural amino acid analog of histidine that can act as a proton shuttle in an enzyme.