Mechanism-Based Allylic Carbasugar Chlorides That Form Covalent Intermediates with α- and β-Galactosidases

Area of Science:

• Biochemistry
• Enzymology
• Medicinal Chemistry

Background:

• Glycoside hydrolases are crucial enzymes involved in various human diseases, including lysosomal storage disorders.
• Developing novel inhibitors for these enzymes is a significant research focus, with many current inhibitors being reversible competitive types.
• Inhibitors often mimic the enzyme's transition state, frequently featuring a protonatable basic nitrogen atom.

Purpose of the Study:

• To investigate novel mechanism-based small molecule inhibitors for glycoside hydrolases.
• To explore the covalent interactions of galacto-like configured cyclohexenyl carbasugars with alpha- and beta-galactosidases.
• To characterize the reaction of Aspergillus oryzae beta-galactosidase with different carbasugar inhibitors.

Main Methods:

• Synthesis and characterization of galacto-like configured cyclohexenyl carbasugars.
• Enzymatic assays to determine the inhibitory activity and covalent complex formation with alpha- and beta-galactosidases.
• Kinetic analysis to determine second-order rate constants (k_inact/K_i) for enzyme-inhibitor reactions.

Main Results:

• Galacto-like configured cyclohexenyl carbasugars were found to form reversible covalent complexes with both alpha-galactosidase and beta-galactosidase.
• Aspergillus oryzae beta-galactosidase reacted with three distinct carbasugar inhibitors, yielding the same enzyme-carbasugar covalent intermediate.
• The alpha-galacto-configured inhibitor was observed to covalently label the beta-galactosidase from Aspergillus oryzae, indicating catalytic versatility.

Conclusions:

• Cyclohexenyl carbasugars represent a novel class of mechanism-based inhibitors that form reversible covalent complexes with galactosidases.
• The unexpected labeling of beta-galactosidase by an alpha-galacto-configured inhibitor underscores the broad catalytic capabilities of glycoside hydrolases.
• These covalent inhibitors are anticipated to become valuable tools in chemical biology for studying enzyme mechanisms and developing therapeutics.