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Volume 272, Number 32, Issue of August 8, 1997 pp. 19731-19737
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Tryptophan Fluorescence Reports Nucleotide-induced Conformational Changes in a Domain of the ArsA ATPase

(Received for publication, February 25, 1997, and in revised form, April 30, 1997)

From the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201

The ars operon of plasmid R773 encodes an ATP-dependent extrusion pump for arsenite and antimonite in Escherichia coli. The ArsA ATPase is the catalytic subunit of the pump protein, with two nucleotide binding consensus sequences, one in the NH₂-terminal half and one in the COOH-terminal half of the protein. A 12-residue consensus sequence (DTAPTGHTIRLL) has been identified in ArsA homologs from eubacteria, archebacteria, fungi, plants, and animals. ArsA enzymes were constructed containing single tryptophan residues at either end of this conserved sequence. The emission spectrum of the fluorescence of the tryptophan on the COOH-terminal end (Trp-159) indicated a relatively hydrophilic environment for this residue. An increase in intrinsic tryptophan fluorescence and a blue shift of the maximum emission wavelength were observed upon addition of MgATP, indicating movement of Trp-159 into a relatively less polar environment. No fluorescence response was observed with MgADP, with nonhydrolyzable ATP analogs, or with MgATP by catalytically inactive enyzmes. This suggests that the location Trp-159 is shifted only during hydrolysis of ATP. In contrast, the emission spectrum of Trp-141, located on the NH₂-terminal side of the consensus sequence, indicated a relatively nonpolar environment. The maximum emission wavelength red shifted upon addition of MgADP. MgATP slowly produced a response that correlated with product formation, suggesting that the environment of Trp-141 is sensitive only to MgADP binding. Thus, during ATP hydrolysis the COOH-terminal end of the conserved domain moves into a less polar environment, whereas the NH₂-terminal end moves into a more hydrophilic environment as product is formed. A hypothesis is presented in which the conserved domain of ArsA and homologs is an energy transduction domain involved in transmission of the energy of ATP hydrolysis to biological functions such as transport.

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