Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of sulfolobus solfataricus.
ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.
Pretz
Monika G
MG
Department of Molecular Microbiology, University of Groningen, Groningen, The Netherlands.
Albers
Sonja-Verena
SV
Schuurman-Wolters
Gea
G
Tampé
Robert
R
Driessen
Arnold J M
AJ
van der Does
Chris
C
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000737:chemistry; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001693:Biological Transport, Active / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D051246:Glucose Transport Proteins, Facilitative / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D020125:Mutation, Missense; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013816:Thermodynamics
D018528:ATP-Binding Cassette Transporters; D019843:Archaeal Proteins; D051246:Glucose Transport Proteins, Facilitative; D000255:Adenosine Triphosphate