Nonequivalence of the Nucleotide Binding Domains of the ArsA ATPase

Yong Jiang,Hiranmoy Bhattacharjee,Tongqing Zhou,Barry P Rosen,Suresh V Ambudkar,Zuben E Sauna

doi:10.1074/jbc.m413391200

Abstract

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5'-[alpha-(32)P]-triphosphate at 4 degrees C indicate that metalloid stimulation correlates with a >10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with (32)P at the gamma position was used to measure hydrolysis of trinucleotide at 37 degrees C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.

Highlights

Introduction of a Thrombin Site inArsA—A thrombin site was introduced in the arsA gene by site-directed mutagenesis using the Altered Sites II in vitro mutagenesis system (Promega)
Comparison of incorporation of 8-azido[␣-32P]ATP and 8-azido[␥-32P]ATP under catalytic conditions indicates whether hydrolysis of the trinucleotide has occurred
There was little difference between the arsenite resistance conferred by the wild type arsA gene and the tharsA gene containing the sequence for the thrombin site (Fig. 4), demonstrating that the gene product is functional in vivo

Summary

Nonequivalence of the Nucleotide Binding Domains of the ArsA ATPase*

To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. The ATP analogue labeled with 32P at the ␥ position was used to measure hydrolysis of trinucleotide at 37 °C Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. Both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2 These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent. The two NBDs have different properties, suggesting that they may have different functions

ArsA Nucleotide Binding Domains

RESULTS

DISCUSSION