Introduction of Histidine Analogs Leads to Enhanced Proton Transfer in Carbonic Anhydrase V
| Autor: | J. N. Earnhardt, Philip J. Laipis, David N. Silverman, Chingkuang Tu, S. K. Wright, Ronald E. Viola, Minzhang Qian |
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| Rok vydání: | 1999 |
| Předmět: |
Stereochemistry
Biophysics Oxygen Isotopes Biochemistry Catalysis Substrate Specificity Mice Residue (chemistry) chemistry.chemical_compound Carbonic anhydrase Animals Imidazole Organic chemistry Histidine Cysteine Molecular Biology Carbonic Anhydrases chemistry.chemical_classification Mice Inbred BALB C biology Leaving group Active site Chemical modification Enzyme chemistry Mutagenesis Site-Directed biology.protein Protons |
| Zdroj: | Archives of Biochemistry and Biophysics. 361:264-270 |
| ISSN: | 0003-9861 |
| DOI: | 10.1006/abbi.1998.0984 |
| Popis: | The rate-limiting step in the catalysis of the hydration of CO2 by 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 pKa near 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. |
| Databáze: | OpenAIRE |
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