Substrate-induced inactivation of a crippled .beta.-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role
| Autor: | Ruedi Aebersold, R. Antony J. Warren, Mark Namchuk, Stephen G. Withers, Donald E. Trimbur, John C. Gebler |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
chemistry.chemical_classification
Base Sequence biology Chemistry Agrobacterium beta-Glucosidase DNA Mutational Analysis Molecular Sequence Data Mutant Substrate (chemistry) biology.organism_classification Biochemistry Substrate Specificity Amino acid Hydrolysis Enzyme Mutagenesis Site-Directed Glycoside hydrolase Amino Acids Tyrosine Sequence Analysis Rhizobium |
| Zdroj: | Biochemistry. 34:14547-14553 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | The beta-glucosidase from Agrobacterium sp. catalyzes the hydrolysis of beta-glucosides via a covalent alpha-D-glucopyranosyl-enzyme intermediate involving Glu358. Hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside by the low activity Glu358Asp mutant of Agrobacterium beta-glucosidase is accompanied by time-dependent inactivation of the enzyme. Through kinetic studies, labeling, and sequence analysis, inactivation is shown to be a consequence of the occasional (1 time in 1100) attack of Tyr298 on the anomeric center of the substrate, in place of the catalytic nucleophile, with formation of a stable alpha-D-glucopyranosyl tyrosine residue. Tyr298 is conserved throughout family 1 of glycoside hydrolases, an indication of a possible role in catalysis. Results of a kinetic analysis of the Tyr298Phe mutant are consistent with a function of Tyr298 in both orienting the nearby nucleophile Glu358 and stabilizing its deprotonated state in the free enzyme. |
| Databáze: | OpenAIRE |
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