Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.
Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.
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Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
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D000048:Acanthamoeba / Q000201:enzymology* / Q000235:genetics; D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019143:Evolution, Molecular; D006868:Hydrolysis; D007700:Kinetics; D009218:Myosins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000199:Actins; D015800:Protozoan Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins