Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101
| Autor: | Martin Willemoës, Anders Lønstrup Hansen, Ramon Crehuet, Signe F. Simonsen, Johanna M Koivisto, Dennis K. Hansen, Zehui Dong |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
chemistry.chemical_classification
Glycan Bifidobacterium longum biology Chemistry Stereochemistry Substrate (chemistry) Peptides and proteins Molecules biology.organism_classification Biochemistry Residue (chemistry) Hydrolysis Enzyme Nucleophile Substitution reactions biology.protein Kinetic parameters Enzyme kinetics |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname Hansen, A L, Koivisto, J M, Simonsen, S, Dong, Z, Crehuet, R, Hansen, D K & Willemoës, M 2021, ' Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101 ', Biochemistry, vol. 60, no. 45, pp. 3398-3407 . https://doi.org/10.1021/acs.biochem.1c00596 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.1c00596 |
| Popis: | Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galβ1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galβ(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile. This work was supported by the Danish Dairy Research Foundation, the Villum Foundation, and the Independent Research Fund Denmark (9041-00126B) to MW. The work has been carried out using CSUC resources (RC). |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|