NDLI: Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

Content Provider	Journal of Biological Chemistry (JBC)
Author	Kaur, P. Rosen, B. P.
Abstract	An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.
Related Links	http://www.jbc.org/content/267/27/19272.abstract
Ending Page	19277
Starting Page	19272
Page Count	6
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	27
Volume Number	267
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1992-09-25
Access Restriction	Open
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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