The Anion-stimulated ATPase ArsA Shows Unisite and Multisite Catalytic Activity

Parjit Kaur

doi:10.1074/jbc.274.36.25849

Abstract

ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.

Highlights

ArsA is an anion-stimulated ATPase that consists of two consensus nucleotide binding sites, one in the N-terminal (A1) and other in the C-terminal (A2) half of the protein [1]
It was proposed that the A1 site is a high affinity ATP site that binds ATP in the absence of the ligand, whereas the A2 site has a very low affinity, and the binding of the ligand, arsenite or antimonite, acts as a switch that allows ATP binding to the A2 site
Allosteric regulation of the ATPase activity of ArsA by antimonite appears to occur by a novel mechanism, in which binding of antimonite acts as an “on/off” switch that allows ATP binding to the A2 site [7]

Summary

Introduction

ArsA is an anion-stimulated ATPase that consists of two consensus nucleotide binding sites, one in the N-terminal (A1) and other in the C-terminal (A2) half of the protein [1]. ATP binding studies carried out between ArsA and [␣-32P]ATP by UV cross-linking showed that only the A1 domain is involved in forming a UV-activated adduct in the presence of ATP [4, 5]. It was proposed that the A1 site is a high affinity ATP site that binds ATP in the absence of the ligand, whereas the A2 site has a very low affinity, and the binding of the ligand, arsenite or antimonite, acts as a switch that allows ATP binding to the A2 site It is not clear whether two sites in ArsA have independent catalytic activity or what is the mechanism for catalytic co-operativity in this protein. The present study shows that the A1 domain of ArsA is capable of carrying out unisite catalysis in the absence of antimonite at a low rate. The data shown here suggest that the A2 site is not an independent site and that it comes into action only after the A1 site is occupied, indicating antimonite-induced interaction between the A1 and A2 sites and providing first biochemical evidence of cooperativity between the the two sites in ArsA

Methods

Results

Conclusion