Kambara Taketoshi, Komaba Shigeru, Ikebe Mitsuo
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
J Biol Chem. 2006 Dec 8;281(49):37291-301. doi: 10.1074/jbc.M603823200. Epub 2006 Oct 1.
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.
肌球蛋白IIIA在光感受器细胞中表达，被认为在光转导过程中起关键作用，但其分子层面的功能在很大程度上尚不清楚。在此，我们阐明了人肌球蛋白IIIA的ATP酶循环的动力学机制。在肌动蛋白浓度极低的情况下，稳态ATP酶活性显著激活，约提高了10倍。肌动球蛋白IIIA中ADP的解离速率比整个循环速率大10倍，因此不是限速步骤。肌动蛋白解离形式的ATP水解步骤的速率常数非常慢，但肌动蛋白结合可显著加速该速率。肌球蛋白IIIA的ATP结合形式与肌动蛋白的解离常数为亚微摩尔级，这与低K（肌动蛋白）值非常吻合。这些结果表明，对于肌动球蛋白IIIA的ATP酶反应，ATP水解主要发生在肌动蛋白结合形式中。所获得的K（肌动蛋白）远低于先前报道的值，我们发现肌球蛋白IIIA的自磷酸化显著增加了K（肌动蛋白），而V（max）不变。我们的动力学模型表明，肌动蛋白附着的水解和无机磷酸（Pi）释放步骤都决定了去磷酸化形式的整体循环速率。尽管肌动球蛋白IIIA的ATP酶反应的稳定稳态中间体不是典型的强肌动蛋白结合中间体，但稳定中间体对肌动蛋白的亲和力远高于传统的弱肌动蛋白结合形式。目前的结果表明，肌球蛋白IIIA在其ATP水解循环的大部分时间里都与肌动蛋白结合。
