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J. Biol. Chem., Vol. 282, Issue 1, 216-231, January 5, 2007

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Kinetic Mechanism of Human Myosin IIIA^*

Andréa C. Dosé, Shobana Ananthanarayanan, Judy E. Moore, Beth Burnside, and Christopher M. Yengo¹

From the Department of Biology, University of North Carolina, Charlotte, North Carolina 28223 and the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k_cat = 0.77 ± 0.08 s^–1), and high actin concentrations are required to fully activate the ATPase rate (K_ATPase = 34 ± 11 µM). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K_actin 7 µM). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K₁k₊₂ = 0.020 and 0.001 µM^–1·s^–1, respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s^–1) than the k_cat, whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s^–1). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K_d 5 µM). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.

Received for publication, June 22, 2006 , and in revised form, October 23, 2006.

^* This work was supported by National Institutes of Health Grant R03EY016419, an American Heart Association scientist development grant (to C. M. Y.), and National Institutes of Health Grant EY03575 (to B. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 704-687-8530; E-mail: cmyengo{at}email.uncc.edu.

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