Decavanadate binding to a high affinity site dear the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity

Decameric vanadate (V-10) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K-i = 0.27 +/- 0.05 muM) in myosin subfragment-1 (S1). The binding of V-10 to S1 can be monitored from titration with V-10 of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-l-naphthyl)-ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V-10 binding site per monomer with a dissociation constant of 0.16-0.7 muM, indicating that S1 labeling with these dyes produced only a small distortion of the V-10 binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V-10 indicated that the V-10 binding site is close to Cys-697 and 707. Fluorescence Studies demonstrated the following: (i) the binding of V-10 to S1 is not competitive either with actin or with ADP(.)V(1) or ADP(.)AlF(4); (ii) the affinity of V-10 for the complex S1/ADP(.)V(1) and S1/ ADP(.)AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand (PP5)-P-1-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V-10 is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V-10 to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.