Binding of fluoride by yeast enolase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein-ligand interactions

Background:

• Yeast enolase is a crucial enzyme in glycolysis.
• Fluoride (F) is known to inhibit enolase activity.
• Understanding the binding mechanism of F is essential for enzyme function studies.

Purpose of the Study:

• To investigate the kinetics and mechanism of fluoride binding to yeast enolase.
• To elucidate the roles of magnesium (Mg2+) and inorganic phosphate (Pi) in fluoride binding.
• To characterize the binding sites and affinities for fluoride on the enolase dimer.

Main Methods:

• Direct measurement of equilibrium fluoride concentrations using an ion-specific electrode.
• Kinetic analysis under varying conditions of Mg2+, Pi, and pH.
• Characterization of enzyme-ligand interactions and quaternary complex formation.

Main Results:

• Fluoride binding is influenced by Mg2+ and Pi, suggesting an ordered binding mechanism.
• Yeast enolase binds a maximum of four fluoride atoms per dimer, with nonequivalent binding affinities.
• Dissociation constants for fluoride pairs were determined (5.0 X 10(-4) M and 8.2 X 10(-5) M).
• Binding of the first fluoride pair involves conformational Mg2+ ions, while the second pair involves catalytic Mg2+ and exhibits positive cooperativity.
• Phosphate binding may involve sequential addition to Mg2+ species.
• Fluoride binding is optimal at pH 5.5-6.0, and 2-phosphoglycerate releases bound fluoride.

Conclusions:

• The study reveals a complex, ordered binding mechanism for fluoride on yeast enolase, involving specific interactions with Mg2+ and Pi.
• The findings provide insights into how fluoride inhibits enolase activity at a molecular level.
• This detailed understanding of fluoride-enolase interactions can inform strategies for enzyme modulation and drug design.