Substrate-induced dimerization of the ArsA protein, the catalytic component of an anion-translocating ATPase
- Wayne State Univ., Detroit, MI (USA)
The ArsA protein, the catalytic component of the plasmid-encoded resistance system for removal of the toxic oxyanions arsenite, antimonite, and arsenate from bacterial cells, catalyzes oxyanion-stimulated ATP hydrolysis. Three lines of evidence suggest that the ArsA protein functions as a homodimer. First, the ArsA protein was modified with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA). Antimonite potentiated FSBA inhibition, while ATP or ADP afforded partial protection. ATP and antimonite together provided complete protection, indicating interaction of the anion- and nucleotide-binding sites. The estimated Ki values for FSBA were 0.4 mM in the absence of antimonite and 0.1 mM in the presence of antimonite. Incorporation of (14C)FSBA was examined. Extrapolation of the amount of FSBA required to inactivate the protein indicated that 1 mol of FSBA was sufficient to inhibit the activity of 1 mol of ArsA protein in the absence of substrates, while only 0.5 mol was required in the presence of the anionic substrate antimonite. Second, chemical cross-linking of the 63-kDa ArsA protein with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline resulted in formation of a species approximately twice the size of the monomer in the presence of antimonite but not ATP. Third, determination of the average mass of the ArsA protein in solution by light scattering demonstrated that the average species was 66 kDa in the absence of substrates. In the presence of antimonite the weight average molecular mass increased to a mass in excess of 100 kDa. These results are consistent with the ArsA protein existing in an equilibrium between monomer and dimer, with the equilibrium favoring dimerization upon binding of the anionic substrate. Moreover, total loss of ATPase activity in the half-modified enzyme suggests that the catalytic sites on each monomer must interact.
- OSTI ID:
- 6057940
- Journal Information:
- Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 266:4; ISSN JBCHA; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ADENOSINE
ADP
ATP-ASE
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CATALYSIS
CHEMICAL REACTIONS
DIMERIZATION
ENZYME ACTIVITY
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MEMBRANE PROTEINS
MOLECULAR WEIGHT
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
POLYMERIZATION
PROTEINS
REACTION KINETICS
REAGENTS
RECEPTORS
RIBOSIDES
SCATTERING
SUBSTRATES
TRACER TECHNIQUES