Kinetic mechanism of human myosin IIIA.
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 microm). 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) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-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) approximately 5 microm). 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.
Dosé
Andréa C
AC
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Ananthanarayanan
Shobana
S
Moore
Judy E
JE
Burnside
Beth
B
Yengo
Christopher M
CM
D000199:Actins / Q000737:chemistry; D020816:Amino Acid Motifs; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D025102:Myosin Type III / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D000199:Actins; D010710:Phosphates; D025102:Myosin Type III; D009218:Myosins