|Title||Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.|
|Publication Type||Journal Article|
|Year of Publication||2003|
|Authors||Lah, N., J. Lah, I. Zegers, L. Wyns, and J. Messens|
|Journal||J Biol Chem|
|Date Published||2003 Jul 4|
|Keywords||Arsenite Transporting ATPases, Calorimetry, Circular Dichroism, Enzyme Stability, Ion Pumps, Multienzyme Complexes, Mutagenesis, Site-Directed, Plasmids, Potassium, Protein Binding, Staphylococcus aureus|
Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.
|Alternate Journal||J. Biol. Chem.|
Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.