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 |
Volume | 278 |
Issue | 27 |
Pagination | 24673-9 |
Date Published | 2003 Jul 4 |
ISSN | 0021-9258 |
Keywords | Arsenite Transporting ATPases, Calorimetry, Circular Dichroism, Enzyme Stability, Ion Pumps, Multienzyme Complexes, Mutagenesis, Site-Directed, Plasmids, Potassium, Protein Binding, Staphylococcus aureus |
Abstract | 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. |
DOI | 10.1074/jbc.M303194200 |
Alternate Journal | J. Biol. Chem. |
PubMed ID | 12682056 |
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