Probing the mechanism of ATP hydrolysis on F-actin using vanadate and the structural analogs of phosphate BeF-3 and A1F-4.
The binding of phosphate analogs to F-ADP-actin filaments and their effect on the dynamics of the polymer have been investigated. Orthovanadate binds to F-actin with the same affinity as phosphate and, at low saturation levels, stabilizes the filament structure in a Pi-like fashion; at higher concentration, it promotes destabilization of the filament. BeF-3 and A1F-4 bind to F-ADP-actin in competition with Pi, with a stoichiometry of 1 mol/mol of F-actin subunit and an affinity 3 orders of magnitude higher than Pi (KD = 2 and 25 microM for BeF-3 and A1F-4, respectively). BeF-3 and A1F-4 mimic Pi in stabilizing F-actin and slow down the rate of actin dissociation from filaments 150-fold. Only 1% of F-ADP-BeF3 subunits provide extensive stabilization of the filament. A quantitative analysis of the stabilization by BeF-3 is proposed. While Pi appears in rapid equilibrium with F-ADP-actin, BeF-3 binds to and dissociates from F-ADP-actin at very slow rates (k+ = 4 M-1 S-1; k = 8.10(-6) S-1). In addition, although functionally similar to the reconstituted F-ADP-Pi species, F-ADP-BeF3 has a different conformation as indicated by the 17% quenching of pyrenyl fluorescence linked to BeF-3 binding. We suggest that BeF-3 may be a good analog of the transition state F-ADP-P* and that Pi release following cleavage of ATP on F-actin might be rate-limited by the isomerization of F-ADP-P*.
Combeau
C
C
Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Carlier
M F
MF
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000535:Aluminum / Q000378:metabolism*; D017607:Aluminum Compounds; D000818:Animals; D001608:Beryllium / Q000378:metabolism*; D001665:Binding Sites; D005459:Fluorides / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009132:Muscles / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D014638:Vanadates / Q000378:metabolism* / Q000494:pharmacology
D000199:Actins; D017607:Aluminum Compounds; D010710:Phosphates; C042164:sodium aluminum tetrafluoride; C056920:beryllium trifluoride; D014638:Vanadates; D000255:Adenosine Triphosphate; D000535:Aluminum; D001608:Beryllium; D005459:Fluorides