title: The kinetic mechanism of mouse myosin VIIA.
abstract: Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s(-1) is activated by actin only by ∼3.5-fold resulting in a V(max) of 0.7 s(-1) and a K(ATPase) of 11.5 μM. There is no single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M·T M·D·P) is slow (12 s(-1)) and the equilibrium constant (K(H)) of 1 suggests significant reversal of hydrolysis. In the presence of actin ADP dissociates with a rate constant of 1.2 s(-1). Phosphate dissociation is relatively fast (>12 s(-1)), but the maximal rate could not be experimentally obtained at actin concentrations ≤ 50 μM because of the weak binding of the myosin VIIa-ADP-P(i) complex to actin. At higher actin concentrations the rate of attached hydrolysis (0.4 s(-1)) becomes significant and partially rate-limiting. Our findings suggest that the myosin VIIa is a "slow", monomeric molecular motor with a duty ratio of 0.6.
authors.affiliation: Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Haithcock
authors.forename: Neil
authors.initials: N
authors.lastname: Billington
authors.forename: Kevin
authors.initials: K
authors.lastname: Choi
authors.forename: Jennifer
authors.initials: J
authors.lastname: Fordham
authors.forename: James R
authors.initials: JR
authors.lastname: Sellers
authors.forename: Walter F
authors.initials: WF
authors.lastname: Stafford
authors.forename: Howard
authors.initials: H
authors.lastname: White
authors.forename: Eva
authors.initials: E
authors.lastname: Forgacs
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D007700:Kinetics; D051379:Mice; D000081412:Myosin VIIa; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: C000631456:Myo7a protein, mouse; D000081412:Myosin VIIa; D000255:Adenosine Triphosphate; D009218:Myosins