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Engineering the enolase magnesium II binding site: implications for its evolution.

Bettina Schreier1, Birte Höcker

  • 1Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.

Biochemistry
|August 10, 2010
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Summary

The second magnesium ion (MgII) is essential for yeast enolase 1 activity. Removing MgII coordinating residues and replacing substrate interactions prevents catalysis, highlighting MgII

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Protein structure-function relationships

Background:

  • Enolase, a glycolytic enzyme, catalyzes the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP).
  • Two magnesium ions (MgI and MgII) in the active site are crucial for enolase catalysis, activating the C2 proton of 2-PGA and stabilizing intermediates.
  • While other enolase superfamily members utilize a single magnesium ion (MgI), the role of MgII in yeast enolase 1 remains less understood.

Purpose of the Study:

  • To investigate the specific role of the second magnesium ion (MgII) in the catalytic mechanism of yeast enolase 1.
  • To determine if MgII is essential for enolase activity or if MgI alone is sufficient under modified conditions.

Main Methods:

  • Site-directed mutagenesis to remove MgII coordinating residues in yeast enolase 1.
  • Introduction of positively charged side chains to replace substrate-MgII interactions.
  • High-resolution X-ray crystallography to determine structural changes.
  • Enzyme activity assays to measure catalytic efficiency.

Main Results:

  • Mutated yeast enolase 1 variants failed to bind MgII, confirming the successful removal of MgII coordinating residues.
  • The introduced positively charged side chains effectively blocked MgII binding but did not restore catalytic activity.
  • Control mutants retained basal enolase activity, indicating MgI-dependent catalysis in the absence of MgII.
  • Precatalytic binding of 2-PGA to the apo state of enolase was observed.

Conclusions:

  • Yeast enolase 1 is inactive without the second magnesium ion (MgII), underscoring its critical role in catalysis.
  • The findings suggest that ancestral enolase activity may have evolved with only MgI-dependent catalysis.
  • MgII is indispensable for the high catalytic efficiency of yeast enolase 1.