Kinetic mechanism and regulation of myosin VI
Kinetic mechanism and regulation of myosin VI
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DOI：
10.1074/jbc.m104136200
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发表时间：
2001-08-24
影响因子：
4.8
通讯作者：
Sweeney, HL
中科院分区：
文献类型：
--
作者：
De la Cruz, EM;Ostap, EM;Sweeney, HL
肌球蛋白VI是唯一被鉴定的指向末端的肌球蛋白，并且可能在体内由肌动蛋白结合位点处的重链磷酸化（HCP）调节。我们对肌动球蛋白VI ATP酶循环进行了详细的动力学分析，以确定是否有独特的适应性来支持反向方向性，并确定HCP调控的分子基础。ADP释放是循环中的限速步骤。ATP结合缓慢，亲和力低。在生理核苷酸浓度下，肌球蛋白VI与肌动蛋白强烈结合，并占据无核苷酸（僵硬）和ADP结合状态。因此，肌球蛋白VI是一个高占空比的马达，适用于保持张力，并有潜力进行。模拟HCP的突变体增加了P-1释放速率，这降低了K-ATP酶，但不影响ADP释放。这些测量是第一次直接测量HCP对任何肌球蛋白调节的步骤。双头肌球蛋白VI的测量表明，头部是不独立的，和本地二聚体水解多个ATP每个扩散遇到一个肌动蛋白丝。我们提出了一个交替网站模型的步进和持续合成的双头高占空比肌球蛋白。
Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of P-i release, which lowers the K-ATPase, but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins.