Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site
| Autor: | Brian W. Matthews, L. H. Weaver, Ryota Kuroki |
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| Rok vydání: | 1999 |
| Předmět: |
Models
Molecular Threonine Glycosylation Glycoside Hydrolases Protein Conformation Stereochemistry Molecular Sequence Data Mutant Protein Engineering chemistry.chemical_compound Hydrolase Bacteriophage T4 Histidine Glycoside hydrolase Amino Acid Sequence Binding site Binding Sites Multidisciplinary biology Substrate (chemistry) Active site Hydrogen Bonding Biological Sciences Kinetics Amino Acid Substitution Carbohydrate Sequence chemistry Mutagenesis Site-Directed biology.protein Muramidase Lysozyme |
| Zdroj: | Proceedings of the National Academy of Sciences. 96:8949-8954 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.96.16.8949 |
| Popis: | In contrast to hen egg-white lysozyme, which retains the β-configuration of the substrate in the product, T4 lysozyme (T4L) is an inverting glycosidase. The substitution Thr-26 → His, however, converts T4L from an inverting to a retaining enzyme. It is shown here that the Thr-26 → His mutant is also a transglycosidase. Indeed, the transglycosylation reaction can be more effective than hydrolysis. In contrast, wild-type T4L has no detectable transglycosidase activity. The results support the prior hypothesis that catalysis by the Thr-26 → His mutant proceeds via a covalent intermediate. Further mutations (Glu-11 → His, Asp-20 → Cys) of the T26H mutant lysozyme indicate that the catalytic mechanism of this mutant requires Glu-11 as a general acid but Asp-20 is not essential. The results help provide an overall rationalization for the activity of glycosidases, in which a highly conserved acid group (Glu-11 in T4L, Glu-35 in hen egg-white lysozyme) on the β-side of the substrate acts as a proton donor, whereas alterations in the placement and chemical identity of residues on the α-side of the substrate can lead to catalysis with or without retention of the configuration, to transglycosidase activity, or to the formation of a stable enzyme-substrate adduct. |
| Databáze: | OpenAIRE |
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