New insights into the structure-function relationships of Rho-associated kinase: a thermodynamic and hydrodynamic study of the dimer-to-monomer transition and its kinetic implications
New insights into the structure-function relationships of Rho-associated kinase: a thermodynamic and hydrodynamic study of the dimer-to-monomer transition and its kinetic implications
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DOI：
10.1042/bj20040344
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发表时间：
2004-12-01
影响因子：
4.1
通讯作者：
Fox, T
中科院分区：
文献类型：
--
作者：
Doran, JD;Liu, X;Fox, T
ROCK（Rho相关激酶）油其寡聚化状态的长度的影响已通过分析全长蛋白质和四个截断的结构，使用光散射和分析超离心方法进行了研究。大小的变化与激酶的动力学性质相关。沉降速度、沉降平衡和光散射数据分析显示，Ser（6）-Arg（415）大小的蛋白质构建体和更大的蛋白质构建体主要以二聚体存在，而较小的构建体主要是单体。包含残基379-415和47-78的氨基酸区段显示为维持二聚体ROCK结构所必需的。以ROCK肽（KKRNRTLSV）和肌球蛋白轻链20 000 Da亚基为底物时，k（cat）值分别为0.7 ~ 2.1 s（-1）和1.0 ~ 5.9 s（-1），表明ROCK寡聚化状态对k（cat）值的影响较小。相对于二聚体构建体，单体构建体的ATP K-m值增加了50-80倍，并且使用特异性竞争性ROCK抑制剂Y-27632的K-i比较也显示出至少120倍的增加，表明ATP结合位点的显著扰动。ROCK肽和肌球蛋白轻链底物的相应Ki值增加1.4-16倍，表明底物结合对ROCK寡聚化状态不太敏感。这些结果表明，ROCK的寡聚化状态可能会影响其激酶活性及其与抑制剂的相互作用，并表明二聚体结构是体内正常功能所必需的。
The effect of the length of ROCK (Rho-associated kinase) oil its oligomerization state has been investigated by analysing full-length protein and four truncated constructs using light-scattering and analytical ultracentrifugation methods. Changes in size correlate with the kinetic properties of the kinase. Sedimentation velocity, sedimentation equilibrium and light-scattering data analyses revealed that protein constructs of size Ser(6)-Arg(415) and larger exist predominantly as dimers, while smaller constructs are predominantly monomeric. The amino acid segments comprising residues 379-415 and 47-78 are shown to be necessary to maintain the dimeric ROCK structure. k(cat) values ranged from 0.7 to 2.1 s(-1) and from 1.0 to 5.9 s(-1) using ROCK peptide (KKRNRTLSV) and the 20 000 Da subunit of myosin light chain respectively as substrate, indicating that the effect of the ROCK oligomerization state on the k(cat) is minor. Values of ATP K-m for monomeric constructs were increased by 50-80-fold relative to the dimeric constructs, and K-i comparisons using the specific competitive ROCK inhibitor Y-27632 also showed increases of at least 120-fold, demonstrating significant perturbations in the ATP binding site. The corresponding K,, values for the ROCK peptide and myosin light chain substrates increased in the range 1.4-16-fold, demonstrating that substrate binding is less sensitive to the ROCK oligomerization state. These results show that the oligomerization state of ROCK may influence both its kinase activity and its interactions with inhibitors, and suggest that the dimeric structure is essential for normal in vivo function.