Kinetics and active site dynamics of Staphylococcusaureus arsenate reductase

被引:0
作者
Joris Messens
José C. Martins
Elke Brosens
Karolien Van Belle
Doris M. Jacobs
Rudolph Willem
Lode Wyns
机构
[1] Dienst Ultrastructuur,
[2] Vlaams interuniversitair Instituut voor Biotechnologie (VIB),undefined
[3] Vrije Universiteit Brussel,undefined
[4] Paardenstraat 65,undefined
[5] 1640 Sint-Genesius-Rode,undefined
[6] High resolution NMR Centre,undefined
[7] Vrije Universiteit Brussel,undefined
[8] Pleinlaan 2,undefined
[9] 1050 Brussels,undefined
来源
JBIC Journal of Biological Inorganic Chemistry | 2002年 / 7卷
关键词
Arsenate reductase Kinetics NMR spectroscopy Redox enzyme Selwyn test;
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摘要
Arsenate reductase (ArsC) encoded by Staphylococcusaureus 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 Vmax 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 µM, kcat/Km=5.2×104 M–1 s–1), while its product, arsenite, was identified as a mixed inhibitor (K*iu=534 µM, K*ic=377 µM).
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页码:146 / 156
页数:10
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