Human myosin III is a motor having an extremely high affinity for actin.
Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.
Kambara
Taketoshi
T
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Komaba
Shigeru
S
Ikebe
Mitsuo
M
D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025102:Myosin Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000199:Actins; D020409:Molecular Motor Proteins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; C491724:MYO3A protein, human; D025102:Myosin Type III; D018995:Myosin Heavy Chains; D009218:Myosins