Role of cysteinyl residues in metalloactivation of the oxyanion-translocating ArsA ATPase.
The ArsA protein, the catalytic subunit of the oxyanion-translocating ATPase responsible for resistance to arsenicals and antimonials in Escherichia coli, is activated by arsenite or antimonite. Activation is associated with dimerization of the ArsA protein. Enzymatic activity was rapidly but reversibly inhibited by the sulfhydryl reagent methyl methanethiosulfonate, suggesting that at least one cysteinyl residue is required for catalytic activity. Each of the four cysteinyl residues in the ArsA protein, Cys26, Cys113, Cys172, and Cys422, were individually changed to seryl residues. The C26S protein had normal properties. Cells expressing the other three mutations lost resistance to arsenite and antimonite. The C113S, C172S, and C422S enzymes each had relatively normal Km values for ATP but reduced affinity for antimonite and arsenite. The Vmax of the activated enzymes ranged from very low for the C113S and C422S enzymes to near normal for the C172S enzyme. These results suggest a mechanism of activation by formation of a tricoordinate complex between Sb(III) or As(III) and the cysteine thiolates 113, 172, and 422.
Bhattacharjee
H
H
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Li
J
J
Ksenzenko
M Y
MY
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000965:Antimony / Q000494:pharmacology; D053501:Arsenite Transporting ATPases; D018053:Arsenites / Q000494:pharmacology; D001483:Base Sequence; D003545:Cysteine; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D016623:Ion Pumps; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017670:Sodium Compounds / Q000494:pharmacology
D018053:Arsenites; D016623:Ion Pumps; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D017670:Sodium Compounds; C061374:antimonite; C017947:sodium arsenite; D000965:Antimony; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D003545:Cysteine