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Journal of Bacteriology, December 2000, p. 6570-6576, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Vanadate-Induced Trapping of Nucleotides by Purified Maltose Transport Complex Requires ATP Hydrolysis

Susan Sharma and Amy L. Davidson*

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030

Received 2 August 2000/Accepted 6 September 2000

The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK2 transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl2 or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.

* Corresponding author. Mailing address: Department of Molecular Virology and Microbiology, MS: BCM 280, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-4552. Fax: (713) 798-7375. E-mail: davidson{at}bcm.tmc.edu.

Journal of Bacteriology, December 2000, p. 6570-6576, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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