Structure-function relationships in an anion-translocating ATPase

The ArsAB ATPase is an efflux pump located in the inner membrane of Escheri chia coli. This transport ATPase confers resistance to arsenite and antimon ite by their extrusion from the cells. The pump is composed of two subunits , the catalytic ArsA subunit and the membrane subunit,ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which ty pically have two groups of six transmembrane-spanning helical segments and tno nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 trans membrane-spanning segments. ArsB contains the substrate translocation pathw ay and is capable of functioning as an anion uniporter. The 63 kDa ArsA pro tein is a substrate-activated ATPase. It has two homologous halves, Al. and A2, which are clearly the result of an ancestral gene duplication and fusi on. Each half has a consensus NBD. The mechanism of allosteric activation o f the ArsA ATPase has been elucidated by a combination of molecular genetic s and biochemical, structural and kinetic analyses, Conformational changes produced by binding of substrates, activator and/or products could be revea led by stopped-flow fluorescence measurements with single-tryptophan deriva tives of ArsA. The results demonstrate that the rate-limiting step in the o verall reaction is a slow isomerization between two conformations of the en zyme. Allosteric activation increases the rate of this isomerization such t hat product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activit y, can undergo similar conformational changes to overcome a rate-limiting s tep. Thus the ArsAB pump is a useful model for elucidating mechanistic aspe cts of the ABC superfamily of transport ATPases.