Interplay Between Ion Binding and Catalysis in the Thioredoxin-coupled Arsenate Reductase Family

• Published in: Journal of molecular biology 2006-07, Vol.360 (4), p.826-838
• Main Authors: Roos, Goedele, Buts, Lieven, Van Belle, Karolien, Brosens, Elke, Geerlings, Paul, Loris, Remy, Wyns, Lode, Messens, Joris
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arsenite Transporting ATPases; Bacillus subtilis; Bacillus subtilis - enzymology; Binding Sites; Catalysis; Ion Pumps - chemistry; Ion Pumps - metabolism; ITC; Kinetics; Lysine - metabolism; Molecular Sequence Data; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed; Mutation - genetics; Potassium - metabolism; Protein Conformation; quantum chemistry; Sequence Alignment; Sodium - metabolism; Staphylococcus aureus; Staphylococcus aureus - enzymology; structure; thermodynamic cycle; Thioredoxins - metabolism; Water - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2006.05.054

In the thioredoxin (Trx)-coupled arsenate reductase family, arsenate reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disulphide cascade between three conserved cysteine residues (Cys10, Cys82 and Cys89). For its activity, Sa_ArsC benefits from the binding of tetrahedral oxyanions in the P-loop active site and from the binding of potassium in a specific cation-binding site. In contrast, the steady-state kinetic parameters of Bs_ArsC are not affected by sulphate or potassium. The commonly occurring mutation of a histidine (H62), located about 6 Å from the potassium-binding site in Sa_ArsC, to a glutamine uncouples the kinetic dependency on potassium. In addition, the binding affinity for potassium is affected by the presence of a lysine (K33) or an aspartic acid (D33) in combination with two negative charges (D30 and E31) on the surface of Trx-coupled arsenate reductases. In the P-loop of the Trx-coupled arsenate reductase family, the peptide bond between Gly12 and Asn13 can adopt two distinct conformations. The unique geometry of the P-loop with Asn13 in β conformation, which is not observed in structurally related LMW PTPases, is stabilized by tetrahedral oxyanions and decreases the p K a value of Cys10 and Cys82. Tetrahedral oxyanions stabilize the P-loop in its catalytically most active form, which might explain the observed increase in k cat value for Sa_ArsC. Therefore, a subtle interplay of potassium and sulphate dictates the kinetics of Trx-coupled arsenate reductases.