The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA.
ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.
Patzlaff
Jason S
JS
Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
van der Heide
Tiemen
T
Poolman
Bert
B
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001622:Betaine / Q000494:pharmacology; D001692:Biological Transport; D002462:Cell Membrane; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry; D006868:Hydrolysis; D013294:Lactococcus lactis / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D011510:Proteolipids / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D011510:Proteolipids; C020485:proteoliposomes; D001622:Betaine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C412743:OpuA protein, Lactococcus lactis; D005998:Glycine