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Magnesium acetate induces a conformational change in Escherichia coli primase
1Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Abstract:
Primase from Escherichia coli is a single-stranded DNA-dependent RNA polymerase. As such, it requires magnesium to carry out catalysis. Limited tryptic digestion was used to probe the conformations of primase as a function of magnesium acetate concentration. In the absence of magnesium, trypsin cleaved primase at three sites. Magnesium acetate induced a conformational change such that one of these sites became inaccessible to trypsin digestion and a new site became trypsin accessible. The conformational change was only induced by Mg(OAc)2 and not MnCl2, CaCl2, NaOAc or LiCl, indicating a clear magnesium acetate-dependent conformational change. The effect was slightly induced by MgSO4 and MgCl2. An allosteric binding model indicates that primase binds at least two magnesiums in a cooperative manner. The data were best fit to a two-state model in which one conformation had a high affinity for magnesium, KR = 83.4 M-1, and the other state had virtually no affinity.
Insights
Magnesium acetate binding induces a conformational change in E. coli primase, affecting enzyme activity. This magnesium-dependent shift suggests primase binds magnesium ions cooperatively.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Primase from Escherichia coli is a single-stranded DNA-dependent RNA polymerase essential for DNA replication.
- Enzyme catalysis often requires specific cofactor ions, such as magnesium, for activity.
Purpose of the Study:
- To investigate the conformational changes in E. coli primase induced by magnesium acetate.
- To understand the role of magnesium ions in primase structure and function.
Main Methods:
- Limited tryptic digestion was employed to probe primase conformations.
- Varying concentrations of magnesium acetate and other divalent cations were used to assess their effects.
Main Results:
- Magnesium acetate binding induced a significant conformational change in primase, altering trypsin cleavage sites.
- This conformational change was specific to magnesium acetate (Mg(OAc)2), with minor effects from MgSO4 and MgCl2.
- Other cations like MnCl2, CaCl2, NaOAc, and LiCl did not induce the conformational change.
Conclusions:
- E. coli primase undergoes a magnesium acetate-dependent conformational change.
- An allosteric binding model suggests cooperative binding of at least two magnesium ions.
- The enzyme exists in at least two states with differing affinities for magnesium.