Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.

TitleSpecific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.
Publication TypeJournal Article
Year of Publication2003
AuthorsLah, N., J. Lah, I. Zegers, L. Wyns, and J. Messens
JournalJ Biol Chem
Date Published2003 Jul 4
KeywordsArsenite 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 JournalJ. Biol. Chem.
PubMed ID12682056
Research group: