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J. Biol. Chem., Vol. 278, Issue 27, 24673-24679, July 4, 2003

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Specific Potassium Binding Stabilizes pI258 Arsenate Reductase from Staphylococcus aureus^*

Nina Lah   ¶, Jurij Lah  , Ingrid Zegers , Lode Wyns  and Joris Messens 

From the Department Ultrastructure, Vlaams interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium and the University of Ljubljana, Faculty of Chemistry and Chemical Technology, Askerceva 5, P. O. Box 537, 1000 Ljubljana, Slovenia

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 K_a of 3.8 x 10³ 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 k_cat/K_M 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.

Received for publication, March 28, 2003

^* This work was supported by Vlaams interuniversitair Instituut voor Biotechnologie and Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a short-term fellowship as a visiting scientist from the FWO. To whom correspondence should be addressed. Tel.: 386-1-2419-114; Fax: 386-1-2419-220; E-mail: nina.lah{at}uni-lj.si.

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