Vanadate-induced trapping of nucleotides by purified maltose transport complex requires ATP hydrolysis.
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 MalFGK(2) 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 MgCl(2) 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.
Sharma
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Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Davidson
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D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D000266:Adenylyl Imidodiphosphate; D014638:Vanadates; D000244:Adenosine Diphosphate; D008320:Maltose; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases