Kinetics of the ATP hydrolysis cycle of the nucleotide-binding domain of Mdl1 studied by a novel site-specific labeling technique.
We have recently proposed a "processive clamp" model for the ATP hydrolysis cycle of the nucleotide-binding domain (NBD) of the mitochondrial ABC transporter Mdl1 (Janas, E., Hofacker, M., Chen, M., Gompf, S., van der Does, C., and Tampé, R. (2003) J. Biol. Chem. 278, 26862-26869). In this model, ATP binding to two monomeric NBDs leads to formation of an NBD dimer that, after hydrolysis of both ATPs, dissociates and releases ADP. Here, we set out to follow the association and dissociation of NBDs using a novel minimally invasive site-specific labeling technique, which provides stable and stoichiometric attachment of fluorophores. The association and dissociation kinetics of the E599Q-NBD dimer upon addition and removal of ATP were determined by fluorescence self-quenching. Remarkably, the rate of ATP hydrolysis of the wild type NBD is determined by the rate of NBD dimerization. In the E599QNBD, however, in which the ATP hydrolysis is 250-fold reduced, the ATP hydrolysis reaction controls dimer dissociation and the overall ATPase cycle. These data explain contradicting observations on the rate-limiting step of various ABC proteins and further demonstrate that dimer formation is an important step in the ATP hydrolysis cycle.
van der Does
Chris
C
Institute of Biochemistry, Biocenter, Goethe-University, Marie-Curie-Strasse 9, D-60439 Frankfurt a.M., Germany.
Presenti
Chiara
C
Schulze
Katrin
K
Dinkelaker
Stephanie
S
Tampé
Robert
R
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019281:Dimerization; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; C490069:MDL1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate