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Mg2+ binding to alkaline phosphatase correlates with slow changes in protein lability
E Dirnbach1, D G Steel, A Gafni
1Biophysics Research Division, Department of Biological Chemistry, and Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Biochemistry
|September 12, 2001
Summary
The binding of magnesium (Mg2+) to Escherichia coli alkaline phosphatase (AP) stabilizes the protein, creating a low-lability form. This Mg2+ binding is crucial for kinetic stability and involves interactions with Zn2+.
Area of Science:
- Biochemistry
- Protein Folding
- Enzymology
Background:
- Escherichia coli alkaline phosphatase (AP) exists in different forms with varying stability.
- Understanding protein metal ion interactions is key to enzyme function and stability.
Purpose of the Study:
- To investigate the role of Zn2+ and Mg2+ in the in vitro refolding and stability of AP.
- To elucidate the mechanism of AP stabilization by metal ions.
Main Methods:
- In vitro refolding of unfolded AP in the presence of Zn2+ and Mg2+.
- Guanidine hydrochloride denaturation kinetics to assess protein lability.
- Metal ion binding experiments to determine the order and effect of addition.
Main Results:
- Refolding AP with both Zn2+ and Mg2+ yields two protein species with distinct denaturation kinetics.
- Mg2+ binding is rate-limiting for the conversion to a stable, low-lability AP form.
- The presence of Zn2+ slows Mg2+ binding, and two Mg2+ ions are required for significant stabilization.
Conclusions:
- Mg2+ binding confers kinetic stability to AP, reducing denaturation rates.
- The stabilization mechanism may involve long-distance intersubunit interactions.
- Mg2+ plays a critical role in the structural and functional properties of AP.