Temperature Dependence of Nucleotide Association and Kinetic Characterization of Myo1b

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Abstract
Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. It is one of the best characterized myosin-I isoforms and appears to have unique biochemical properties tuned for tension sensing or tension maintenance. We determined the key biochemical rate constants that define the actomyo1b ATPase cycle at 37 °C and measured the temperature dependence of ATP binding, ADP release, and the transition from a nucleotide-inaccessible state to a nucleotide-accessible state (kα). The rate of ATP binding is highly temperature sensitive, with an Arrhenius activation energy 2−3-fold greater than other characterized myosins (e.g., myosin-II and myosin-V). ATP hydrolysis is fast, and phosphate release is slow and rate limiting with an actin dependence that is nearly identical to the steady-state ATPase parameters (Vmax and KATPase). ADP release is not as temperature dependent as ATP binding. The rates and temperature dependence of ADP release are similar to kα suggesting that a similar structural change is responsible for both transitions. We calculate a duty ratio of 0.08 based on the biochemical kinetics. However, this duty ratio is likely to be highly sensitive to strain.