Related Experiment Videos
Yeast enolase carboxyl modification using Woodward's reagent K
Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
|October 1, 1986
Summary
Yeast enolase inactivation by Woodward's reagent K is prevented by metal ions. This suggests metal ions bind to Glu-181, causing protein contraction and protecting carboxyl groups.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Yeast enolase is a key glycolytic enzyme.
- Woodward's reagent K is a carbodiimide used for carboxyl group modification.
Purpose of the Study:
- To investigate the mechanism of yeast enolase inactivation by Woodward's reagent K.
- To determine the protective effect of "conformational" metal ions on enolase activity.
Main Methods:
- Enzyme kinetics assays to measure enolase activity.
- Chemical modification using Woodward's reagent K.
- Peptide mapping after tryptic digestion.
Main Results:
- Woodward's reagent K inactivates yeast enolase by modifying carboxyl groups.
- Binding of one "conformational" metal ion per subunit significantly protects enolase from inactivation.
- Metal ion binding correlates with modification of 13 carboxyl groups/subunit without metal and 17 with metal.
- Ten tryptic peptides are labeled, primarily in the C-terminal half.
- Metal ion binding appears to involve direct coordination to Glu-181 and induces protein contraction.
Conclusions:
- Metal ion binding protects yeast enolase by directly interacting with Glu-181 and inducing conformational changes.
- These conformational changes likely shield other carboxyl groups from modification, preserving enzyme activity.