Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 49, Pages 37291-37301Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M603823200
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- NIAMS NIH HHS [AR048898, AR04856] Funding Source: Medline
- NIDCD NIH HHS [DC006103] Funding Source: Medline
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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 similar to 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.
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