The mechanism of the reverse recovery step, phosphate release, and actin activation of Dictyostelium myosin II.
The rate-limiting step of the myosin basal ATPase (i.e. in absence of actin) is assumed to be a post-hydrolysis swinging of the lever arm (reverse recovery step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever arm motion, two single tryptophan-containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery step, which is a slow equilibrium step (k(forward) = 0.05 s(-1), k(reverse) = 0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery step, which becomes practically irreversible in the actin-bound form, triggering the power stroke. Even at low actin concentrations the power stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power stroke conformation.
Gyimesi
Máté
M
Department of Biochemistry, Institute of Biology, Eötvös University, Pázmány Péter Sétány 1/A, Budapest, Hungary.
Kintses
Bálint
B
Bodor
Andrea
A
Perczel
András
A
Fischer
Stefan
S
Bagshaw
Clive R
CR
Málnási-Csizmadia
András
A
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002021:Buffers; D018360:Crystallography, X-Ray; D004023:Dictyostelium / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D024462:Myosin Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000199:Actins; D002021:Buffers; D010710:Phosphates; D000244:Adenosine Diphosphate; D024462:Myosin Type II