Structure-function analysis of the ArsA ATPase: contribution of histidine residues.
The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance. Either antimonite or arsenite stimulates ArsA ATPase activity. In this study, the role of histidine residues in ArsA activity was investigated. Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity. The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues. ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site. ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558. Each histidine was individually altered to alanine by site-directed mutagenesis. Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite. The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group.
Bhattacharjee
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Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. hbhattac@med.wayne.edu
Rosen
B P
BP
D000965:Antimony / Q000494:pharmacology; D053501:Arsenite Transporting ATPases; D018053:Arsenites / Q000494:pharmacology; D001483:Base Sequence; D020134:Catalytic Domain; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006639:Histidine / Q000502:physiology*; D016623:Ion Pumps / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D018053:Arsenites; D016623:Ion Pumps; D009097:Multienzyme Complexes; C061374:antimonite; D006639:Histidine; D000965:Antimony; D053501:Arsenite Transporting ATPases; D004047:Diethyl Pyrocarbonate; C015001:arsenite