The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration.
The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration.
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DOI：
10.1016/j.ijbiomac.2009.03.001
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发表时间：
2009-06
影响因子：
8.2
通讯作者：
K. Uda;A. Kuwasaki;Kanami Shima;Tamotsu Matsumoto;Tomohiko Suzuki
中科院分区：
文献类型：
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作者：
K. Uda;A. Kuwasaki;Kanami Shima;Tamotsu Matsumoto;Tomohiko Suzuki
在肌酸激酶(CKs)中，-96氨基酸残基是一种严格保守的精氨酸。该残基与底物结合没有直接关系，但它位于底物肌酸结合部位附近。另一方面，已知的残基-96参与了各种其他磷酸(Guanidino)酶的底物特异性的表达，因为每个酶在这个位置都有一个特定的残基：精氨酸激酶(Tyr)、糖氨酸激酶(ILE)、牛磺酸氨酸激酶(His)和伦布利星激酶(Lys)。为了更好地了解-96残基在CKs中的作用，我们用其他19个氨基酸替换斑马鱼Danio rerio细胞质CK中的这一残基，并在大肠杆菌中表达了这些结构。包括野生型在内的20种重组酶均以可溶性形式获得，并对其活性进行了正向测定。与野生型相比，R96K突变体的活性显著(野生型的8.3%)，而10个突变体(R96Y、A、S、E、H、T、F、C、V和N)的活性较弱(0.056-1.0%)。在其余的突变体(R96Q、G、M、P、L、W、D和I)中，酶活性低于0.05%。我们的诱变研究表明，Danio CK中的Arg-96可以被Lys部分替代，但其他替代物会导致活性显著丧失。通过对鱼雷胞质CK的晶体结构(过渡态和开态)的仔细考察，我们发现R96的侧链只与A339和D340在TSAC结构中形成氢键。基于CKS由四个动态结构域(结构域1-3和固定结构域)组成的假设，上述氢键连接了TSAC结构中可能的结构域1和3。我们认为，CK中的残基-96和其他磷酸原激酶中的等量残基具有双重作用：(1)一个参与区分胍底物；(2)另一个在组织残基-96周围的氢键网络中发挥关键作用，为高催化转换率提供合适的活性中心。该网络的发展模式似乎是独一无二的每个磷酸原激酶，反映了每个酶的进化。
In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.