Circular dichroism (CD) studies on yeast enolase: activation by divalent cations

Area of Science:

• Biochemistry
• Enzyme kinetics
• Structural biology

Background:

• Yeast enolase is a key enzyme in glycolysis.
• Understanding enzyme structure-function relationships is crucial for drug development.

Purpose of the Study:

• To investigate the effects of divalent cations and substrates on yeast enolase structure.
• To identify cation binding sites and their impact on enzyme conformation.

Main Methods:

• Circular dichroism (CD) spectroscopy in the near and far ultraviolet regions.
• Visible absorption spectroscopy.
• Enzyme inhibition studies.

Main Results:

• Divalent cations (Ca2+, Mg2+, Ni2+) induced identical changes in the near-UV CD spectrum, suggesting binding to the same sites and altering tertiary structure.
• Magnesium ions did not affect the far-UV spectrum, indicating no change in secondary structure.
• Substrate binding induced CD spectral changes that were nearly the reverse of those caused by metal ions.
• A competitive inhibitor lacking carbon-3 did not affect the CD spectrum, implying carbon-3 is essential for spectral changes.
• Spectroscopic analysis of Ni2+ and Co2+ binding indicated octahedral coordination with oxyligands.
• Substrates and transition state analogues perturbed conformational sites, while catalytic and inhibitory sites showed minimal CD activity.

Conclusions:

• Metal ion binding significantly alters yeast enolase's tertiary structure at specific sites.
• The enzyme's secondary structure remains unaffected by magnesium ions.
• Carbon-3 is critical for cation-induced spectral changes.
• Distinct conformational, catalytic, and inhibitory sites exist on the enzyme, with varying CD activity.