Reactive thioglucoside substrates for β-glucosidase

Area of Science:

• Biochemistry
• Enzymology
• Glycoscience

Background:

• Thioglycosides are generally resistant to hydrolysis by acids and glycohydrolases.
• β-glucosidases are crucial enzymes involved in carbohydrate metabolism.
• Understanding enzyme-substrate interactions is key to developing novel biocatalysts.

Purpose of the Study:

• To identify and characterize novel, efficient thioglycoside substrates for β-glucosidase.
• To elucidate the mechanism of hydrolysis for these new substrates.
• To compare the substrate efficiency and reaction mechanism with existing substrates.

Main Methods:

• Synthesis of β-D-glucopyranosides of 2-mercaptobenzimidazole (GlcSBiz) and 2-mercaptobenzoxazole (GlcSBox).
• Enzymatic hydrolysis assays using β-glucosidase from sweet almond (Family 1) and Aspergillus niger (Family 3).
• Kinetic studies including solvent deuterium kinetic isotope effect (SKIE) measurements at varying pL.

Main Results:

• GlcSBiz and GlcSBox were found to be excellent substrates for both sweet almond and Aspergillus niger β-glucosidases, with high catalytic efficiency comparable to p-nitrophenyl β-D-glucoside.
• Enzyme-catalyzed hydrolysis of GlcSBiz proceeds with retention of configuration.
• A modest solvent deuterium kinetic isotope effect on kcat was observed for GlcSBiz hydrolysis, suggesting a mechanism involving remote site protonation and subsequent thioglucosidic bond cleavage.

Conclusions:

• The β-D-glucopyranosides of 2-mercaptobenzimidazole and 2-mercaptobenzoxazole represent a new class of highly efficient β-glucosidase substrates.
• The hydrolysis mechanism involves protonation at the ring nitrogen followed by cleavage of the thioglucosidic bond, leading to a thione product.
• These findings offer insights into the catalytic mechanisms of β-glucosidases and potential applications in glycoscience.