Reactive Thioglucoside Substrates for β-Glucosidase
| Autor: | Alexei V. Demchenko, Gary S. Howarth, Scott J. Hasty, Larry D. Byers, Archana R. Parameswar, Elizabeth Alverson-Banks Avegno |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Binding Sites
biology Chemistry Stereochemistry Beta-glucosidase Thioglucosides beta-Glucosidase Aspergillus niger Biophysics Protonation biology.organism_classification Biochemistry Article law.invention Substrate Specificity Solvent Enzyme Activation Hydrolysis law Kinetic isotope effect Enzyme Stability Enzyme kinetics Molecular Biology Walden inversion Protein Binding |
| Popis: | A new, very efficient, class of thioglycoside substrates has been found for β-glucosidase. While thioglycosides are usually resistant to hydrolysis, even in the presence of acids or most glycohydrolases, the β-D-glucopyranosides of 2- mercaptobenzimidazole (GlcSBiz) and 2-mercaptobenzoxazole (GlcSBox) have been found to be excellent substrates for β-glucosidase from both sweet almond (a family 1 glycohydrolase) and Aspergillus niger (a family 3 glycohydrolase), reacting nearly as well as p-nitrophenyl β-D-glucoside. The enzyme-catalyzed hydrolysis of GlcSBiz proceeds with retention of configuration. As with the (1000-fold slower) hydrolysis of phenyl thioglucosides catalyzed by the almond enzyme, the pL (pH/pD)-independent kcat/KM does not show a detectable solvent deuterium kinetic isotope effect (SKIE), but unlike the hydrolysis of phenyl thioglucosides, a modest SKIE is seen on kcat [D2Okcat = 1.28 (±0.06)] at the pL optimum (5.5 ≤ pL ≤ 6.6). A solvent isotope effect is also seen on the KM for the N-methyl analog of GlcSBiz. These results suggest that the mechanism for the hydrolysis of the β-thioglucoside of 2-mercaptobenzimidazole and of 2- mercaptobenzoxazole involves remote site protonation (at the ring nitrogen) followed by cleavage of the thioglucosidic bond resulting in the thione product. |
| Databáze: | OpenAIRE |
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