The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure.
The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure.
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DOI：
10.1016/j.ijbiomac.2006.02.016
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发表时间：
2006-05
影响因子：
8.2
通讯作者：
Jian-wei Zhang;Tong-Jin Zhao;Shi-lei Wang;Qin Guo;Tao-tao Liu;Feng Zhao;Xi-cheng Wang
中科院分区：
文献类型：
--
作者：
Jian-wei Zhang;Tong-Jin Zhao;Shi-lei Wang;Qin Guo;Tao-tao Liu;Feng Zhao;Xi-cheng Wang
精氨酸激酶（Arginine kinase，AK）在无脊椎动物中催化精氨酸被MgATP可逆磷酸化，并与MgADP正向反应生成高能化合物磷酸精氨酸（phosphoarginine，Parg）。探讨海参-AK（二聚体）和鲎-AK（单体）与电鳐肌酸激酶催化作用的不同机制为了揭示二聚体AK的C-末端结构域环（C-环）的结构作用，构建了六个单位点突变体E314 D、E314 Q、E314 V、F315 A、F315 H和F315 Y以及两个多位点变体，S312 R/F315 H/V319 E（通过用单体AK的C环取代二聚体AK的C环而形成，称为AA环）和S312 G/E314 V/F315 D/E317 A/S318 A/G321 S（通过用二聚体CK的C环取代二聚体AK的C环而形成，称为AC环）。Glu 314突变体的AK活性显著降低，从60- 500倍。与Limulus-AK中的相同结构不同，ACloop仅显示出轻微的AK活性。此外，所有Phe 315突变体，包括保留Glu 314的AAloop，都具有适度的AK活性（野生型的5-84%）。上述结果表明，Glu 314在AK二聚体中的催化作用比在单体中更显著。此外，ANS图谱显示，与野生型AK相比，3个Glu 314突变体对变性剂的耐受性略有下降。虽然单体AK在315位有一个His残基，但突变体F315 H和AAloop不能抵抗任何变性剂的扰动，突变体的吉布斯自由能比野生型AK低约2.7kJ/mol。因此，Phe 315在二聚体AK中的作用与His 315在单体AK中的作用不同。这可能有助于稳定天然构象，而鲎AK中的His 315直接与精氨酸的羧酸酯结合。综合以上结果，我们提出了二聚体AK不同于单体AK和二聚体CK的独特机制。
Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by MgATP to form a high-energy compound phosphoarginine (Parg) and MgADP in forward reaction in invertebrates. To detect the different catalytical mechanisms among Stichopus-AK (dimer) and Limulus-AK (monomer) and Torpedo creatine kinase (dimeric CK) and to reveal the structural role of the C-terminal domain loop (C-loop) of dimeric AK, six single-site mutants, E314D, E314Q, E314V, F315A, F315H and F315Y were constructed as well as two multi-site variants, S312R/F315H/V319E (formed by substituting the C-loop of monomeric AK for that of dimeric AK, termed the AAloop) and S312G/E314V/F315D/E317A/S318A/G321S (formed by substituting the C-loop of dimeric CK for that of dimeric AK, termed the ACloop). The AK activity of the three mutants at Glu314decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe315mutants including the AAloop which retained Glu314had modest AK activity (5–84% of the wild type). All the results above suggested that Glu314played a more significant role in catalysis in dimeric AK than in the monomer. In addition, ANS profiles indicated that the tolerance of the three Glu314mutants to denaturant decreased slightly compared with wild type AK. Though monomeric AK has a His residue at site 315, mutants F315H and the AAloop could not resist any perturbation of denaturant, and the mutants showed a Gibbs free energy of about 2.7kJ/mol lower than wild type AK. Therefore Phe315in dimeric AK has a different role from His315in monomeric AK. This might contribute to the stabilization of the native conformation, while His315in Limulus AK directly binded to the carboxylate of arginine. Taking all the results above together, we suggested a unique mechanism in dimeric AK, different from both monomeric AK and dimeric CK.