The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175
| Autor: | Gerard J. Kleywegt, Anu Koivula, A. Vasella, D. Becker, Laura Ruohonen, M.L. Sinnott, M. Weber, K. Piens, M. Szardenings, M. Claeyssens, Tuula T. Teeri, Jerry Ståhlberg, J.Y. Zou, Tapani Reinikainen, T.A. Jones, Gerd Wohlfahrt |
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| Jazyk: | angličtina |
| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular beta-cellobiosyl fluorides Stereochemistry Protein Conformation Molecular Sequence Data Protonation Crystallography X-Ray fusca endocellulase E2 Biochemistry Catalysis Hydrolysis Scissile bond Colloid and Surface Chemistry Cellulase alpha-cellobiosyl Aspartic acid angstrom resolution Cellulose 1 4-beta-Cellobiosidase catalytic core Trichoderma reesei chemistry.chemical_classification Trichoderma Aspartic Acid Binding Sites biology Chemistry Hydrogen bond crystalline cellulose degradation humicola-insolens Active site Glycosidic bond endoglucanase-I General Chemistry biology.organism_classification Carbohydrate Sequence hydrolysis family-6 biology.protein Mutagenesis Site-Directed |
| Zdroj: | Koivula, A, Ruohonen, L, Wohlfahrt, G, Reinikainen, T, Teeri, T, Piens, K, Claeyssens, M, Weber, M, Vasella, A, Becker, D, Sinnott, M L, Zou, J Y, Kleywegt, G J, Szardenings, M, Ståhlberg, J & Jones, T A 2002, ' The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175 ', Journal of the American Chemical Society, vol. 124, no. 34, pp. 10015-10024 . https://doi.org/10.1021/ja012659q |
| DOI: | 10.1021/ja012659q |
| Popis: | Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of D2O(V) of 1.16 ± 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis. |
| Databáze: | OpenAIRE |
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