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External yeast beta-fructosidase. Affinity labeling of the active site
Biochimica Et Biophysica Acta
|December 8, 1976
Summary
Conduritol-B-epoxide irreversibly inhibits yeast beta-fructosidase by binding to its active site. Substrate presence prevents this inactivation, suggesting a specific interaction with the enzyme
Area of Science:
- Biochemistry
- Enzymology
- Enzyme kinetics
Background:
- External yeast beta-fructosidase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) plays a key role in carbohydrate metabolism.
- Understanding enzyme inhibition mechanisms is crucial for drug development and biochemical research.
Purpose of the Study:
- To investigate the mechanism of action of conduritol-B-epoxide as an inhibitor of yeast beta-fructosidase.
- To determine the kinetic parameters and identify the reactive site involved in the inhibition process.
Main Methods:
- Enzyme kinetics studies were performed to analyze the inhibition.
- The pH-dependence of the inactivation process was investigated.
- Structure-activity relationships were inferred based on inhibitor and substrate similarity.
Main Results:
- Conduritol-B-epoxide acts as an active-site directed, irreversible inhibitor of yeast beta-fructosidase.
- The inactivation follows first-order kinetics with respect to time and inhibitor concentration.
- One molecule of inhibitor reacts with one molecule of enzyme.
- Substrate presence protects the enzyme from inactivation.
- pH-dependence analysis revealed two dissociating groups with pKa values of 3.05 and 6.8 involved in inactivation.
Conclusions:
- A carboxylate group at the active site with a pKa of 3.05 is proposed as the reactive site for conduritol-B-epoxide.
- The findings provide insights into the catalytic mechanism and active site structure of yeast beta-fructosidase.
