Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase.
Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM).
Messens
Joris
J
Dienst Ultrastructuur, Vlaams interuniversitair Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel, Paardenstraat 65, 1640 Sint-Genesius-Rode, Belgium. jmessens@vub.ac.be
Martins
José C
JC
Brosens
Elke
E
Van Belle
Karolien
K
Jacobs
Doris M
DM
Willem
Rudolph
R
Wyns
Lode
L
D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism*; D001149:Arsenates / Q000378:metabolism*; D053501:Arsenite Transporting ATPases; D018053:Arsenites / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D016623:Ion Pumps / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
D000838:Anions; D001149:Arsenates; D018053:Arsenites; D004791:Enzyme Inhibitors; D016623:Ion Pumps; D009097:Multienzyme Complexes; D053501:Arsenite Transporting ATPases; C015001:arsenite; C025657:arsenic acid