Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases
| Autor: | Fahimeh S. Shidmoossavee, Saeideh Shamsi Kazem Abadi, Chloe A. N. Gerak, Natalia Sannikova, Andrew J. Bennet, Dustin T. King, Andrew R. Lewis |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Glycoside Hydrolases Stereochemistry Molecular Conformation Biochemistry Cofactor 03 medical and health sciences Hydrolysis Kinetic isotope effect Glycoside hydrolase biology Chemistry Leaving group Galactose NAD biology.organism_classification Citrobacter freundii Kinetics 030104 developmental biology Deuterium 13. Climate action Biocatalysis biology.protein NAD+ kinase |
| Zdroj: | Biochemistry. 57:3378-3386 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.8b00117 |
| Popis: | The glycoside hydrolase family 4 (GH4) α-galactosidase from Citrobacter freundii (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD+ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived βlg value on V/K for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr2+, Y3+, and Mn2+, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured DV/K KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-gal... |
| Databáze: | OpenAIRE |
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