Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Sarah M Heissler,Edward D Korn,Xiong Liu,James R Sellers

doi:10.1074/jbc.m113.485946

Sarah M Heissler, Edward D Korn + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m113.485946

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Abstract

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Highlights

Introduction of theS639D mutation led to a 3-fold increase in the rate of ADP binding compared to WT but had only a minimal effect on the ADP release kinetics (Table 2)
S1 fragments of mammalian nonmuscle myosin-2A, -2B, and -2C had a weak apparent affinity for actin, and actin titration curves did not saturate under the actin concentrations used in those studies [13,14,15]
The crucial function of loop-2 is the formation of the primary actin-myosin interface that is essential for actin-activation of the basal myosin ATPase activity

Summary

Background

Actin-activated ATPase activity of Acanthamoeba myosin-2 is inhibited by phosphorylation of Ser-639. Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 The kinetic parameter predominantly affected by the S639D mutation is the actinactivated release of inorganic phosphate from the acto myosin1⁄7 ADP1⁄7Pi complex, which is the rate-limiting step in the steadystate actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. Structural studies showed that, as for other class-2 myosins, the tail domains of two adjacent heavy chains form a coiled-coil and

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EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION