Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics*

Background: KIF3AB is a heterotrimeric plus-end-directed kinesin-2 motor, implicated in intraflagellar transport. Results: KIF3AB shows rate-limiting ADP release upon microtubule collision and an unusual apparent weak ATP affinity observed at microtubuleKIF3AB dissociation. Conclusion: The presteady-state kinetics suggest a novel ATPase mechanism for KIF3AB stepping. Significance: The KIF3AB kinetics reveal the mechanistic diversity that kinesin motors exhibit for cargo transport. KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 m(-1) s(-1), followed by rate-limiting ADP release at 12.8 s(-1). ATP binding at 7.5 m(-1) s(-1) was followed by an ATP-promoted isomerization at 84 s(-1) to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s(-1). ATP hydrolysis was required for dissociation of the microtubuleKIF3AB complex, which was observed at 22 s(-1). The dissociation step showed an apparent affinity for ATP that was very weak (K-1/2,K-ATP at 133 m). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 m(-1) s(-1), which is inconsistent with fast ATP binding at 7.5 m(-1) s(-1) and a K-d,K-ATP at 6.1 m. These results suggest that ATP binding per se cannot account for the apparent weak K-1/2,K-ATP at 133 m. The steady-state ATPase K-m,K-ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.