Decavanadate binding to a high affinity site near 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 microM) 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-1-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 microM, 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 P(1)P(5)-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.
Tiago
Teresa
T
Departamento de Química e Bioquímica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 8000 Faro, Portugal. ttiago@ualg.pt
Aureliano
Manuel
M
Gutiérrez-Merino
Carlos
C
D000199:Actins / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D020134:Catalytic Domain / Q000187:drug effects; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008027:Light; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000494:pharmacology; D009942:Organometallic Compounds / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D012542:Scattering, Radiation; D014638:Vanadates / Q000378:metabolism* / Q000494:pharmacology
D000199:Actins; D015226:Dinucleoside Phosphates; D004791:Enzyme Inhibitors; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D009942:Organometallic Compounds; C105164:adenosine diphosphate tetrafluoroaluminate; D014638:Vanadates; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000244:Adenosine Diphosphate; C006421:1,5-I-AEDANS; D009218:Myosins