Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10767614 | Biochemical and Biophysical Research Communications | 2007 | 5 Pages |
Abstract
It is shown here that Escherichia coli β-galactosidase has a second Mg2+ binding site that is important for activity. Binding of Mg2+ to the second site caused the kcat (with oNPG as the substrate) to increase about 100 sâ1; the Km was not affected. The Kd for binding the second Mg2+ is about 10â4 M. Since the concentration of free Mg2+ in E. coli is about 1-2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the kcat increase. This indicates that the second Mg2+ site is near to Glu-797. The Ki values of transition state analogs were decreased by small but statistically significant amounts when the second Mg2+ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg2+ helps to order the active site for stabilization of the transition state.
Keywords
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Gopinath Sutendra, Sean Wong, Marie E. Fraser, Reuben E. Huber,