Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain
Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain
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DOI：
10.1021/bi972851y
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发表时间：
1998-05-05
期刊：
影响因子：
2.9
通讯作者：
Manstein, DJ
中科院分区：
文献类型：
--
作者：
Furch, M;Geeves, MA;Manstein, DJ
肌球蛋白的肌动蛋白结合表面环（环2）的突变的影响进行了描述。环2的一部分，肌球蛋白残基618和622之间的片段，被替换为通过引入带正电荷的GKK或中性GNN基序而扩大的序列。产生具有携带多达20个额外氨基酸的环和-1至+12的电荷变化的构建体。使用稳态和瞬时动力学来表征突变马达结构域的酶行为。核苷酸的结合不受环2中的任何改变的影响。关于它们与肌动蛋白的相互作用，具有中等电荷变化（-1至+2）的构建体显示野生型样行为。引入一个以上的GKK基序导致肌球蛋白的肌动蛋白和核苷酸结合位点之间的更强的偶联，并且在不存在ATP和零离子强度下对肌动蛋白的亲和力增加高达1000倍。与野生型构建体M765相比，具有4-12个额外电荷的构建体在它们与肌动蛋白的相互作用中显示出对离子强度的增加的依赖性，k（cat）增加2-3倍，肌动蛋白的K-app减少10倍以上，催化效率增加34-70倍。
The effects of mutations in an actin-binding surface loop of myosin (loop 2) are described. Part of loop 2, the segment between myosin residues 618 and 622, was replaced with sequences enlarged by the introduction of positively charged GKK or neutral GNN motifs. Constructs with loops carrying up to 20 additional amino acids and charge variations from -1 to +12 were produced. Steady-state and transient kinetics were used to characterize the enzymatic behavior of the mutant motor domains. Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with moderate charge changes (-1 to +2) displayed wild-type-like behavior. Introduction of more than one GKK motif led to stronger coupling between the actin-and nucleotide-binding sites of myosin and an up to 1000-fold increased affinity for actin in the absence of ATP and at zero ionic strength, In comparison to the wild-type construct M765, constructs with 4-12 extra charges displayed an increased dependence on ionic strength in their interaction with actin, a 2-3-fold increase in k(cat), a more than 10-fold reduction in K-app for actin, and a 34-70-fold increase in catalytic efficiency.