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Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase
Publication Type:
Journal ArticleSource:
J. Biol. Inorg. Chem., Volume 7, Issue 1-2, p.146 - 156 (2002)URL:
PM:11862551Keywords:
Amino Acid Sequence; Anions; metabolism; Arsenates; Arsenite Transporting ATPases; Arsenites; pharmacology; Binding Sites; physiology; Enzyme Inhibitors; Enzyme Stability; Ion Pumps; antagonists & inhibitors; chemistry; Kinetics; Magnetic Resonance SpectrAbstract:
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)
Notes:
DA - 20020225
IS - 0949-8257 (Print)
LA - eng
PT - Journal Article
PT - Research Support, Non-U.S. Gov't
RN - 0 (Anions)
RN - 0 (Arsenates)
RN - 0 (Arsenites)
RN - 0 (Enzyme Inhibitors)
RN - 0 (Ion Pumps)
RN - 0 (Multienzyme Complexes)
RN - 15502-74-6 (arsenite)
RN - 7778-39-4 (arsenic acid)
RN - EC 3.6.3.16 (Arsenite Transporting ATPases)
SB - IM