Sequence-Specific Ni(II)-Dependent Peptide Bond Hydrolysis for Protein Engineering: Reaction Conditions and Molecular Mechanism
Sequence-Specific Ni(II)-Dependent Peptide Bond Hydrolysis for Protein Engineering: Reaction Conditions and Molecular Mechanism
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DOI：
10.1021/ic1005709
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发表时间：
2010-07-19
影响因子：
4.6
通讯作者：
Bal, Wojciech
中科院分区：
文献类型：
--
作者：
Kopera, Edyta;Krezel, Artur;Bal, Wojciech
最近，我们筛选了一个R-1-（Ser/Thr）-Xaa-His-Zaa-R-2肽的组合文库（Xaa = 17种常见的α-氨基酸，除了Asp、Glu和Cys; Zaa =19种常见的α-氨基酸，除了Cys; R-1 = CH 3CO-Gly-Ala，R-2 = Lys-Phe-Leu-NH 2）并建立了选择Ser/Thr、Xaa、和在Ni（II）离子存在下对特异性R-1-Ser/Thr肽键水解最佳的Zaa取代（Krezel，A.; Kopera，E.; Protas，A. M.; Poznanski，J.; Wysouch-Cieszynska，A.; Bal，W. J. Am. 2010，132，3355 -3366）。通过对七种肽的水解动力学研究证实了筛选结果：R-1-Ser-Arg-His-Trp-R-2、R-1-Ser-Lys-His-Trp-R-2、R-1-Ser-Ala-His-Trp-R-2、R-1-Ser-Arg-His-Ala-R-2、R-1-Ser-Gly-His-Ala-R-2、R-1-Thr-Arg-His-Trp-R-2和R-1-Thr-His-His-Trp-R-2。在本文中，我们使用相同的七个肽，探讨水解反应的分子机理。我们研究了在24和75摄氏度之间的温度下的反应速率的温度依赖性，测量的稳定常数与水解底物和产物的Ni（II）络合物，并研究了在广泛的条件下的R-1-Ser-Arg-His-Trp-R-2肽水解的过程。我们建立了特定的正方形平面复合物含有Ni（II）离子键合到His咪唑氮和三个前面的肽键氮（4 N复合物）是需要的反应发生。反应机理包括N-O酰基转移，产生RI与Ser/Thr羟基的中间体酯。该酯自发水解，产生最终产物。Ni（II）离子通过直接通过肽氮配位使R-1-Ser肽键不稳定以及间接通过在肽链中施加应变来激活R-1-Ser肽键。
Recently we screened a combinatorial library of R-1-(Ser/Thr)-Xaa-His-Zaa-R-2 peptides (Xaa = 17 common alpha-amino acids, except Asp, Glu, and Cys; Zaa =19 common alpha=amino acids, except Cys; R-1 = CH3CO-Gly-Ala, R-2 = Lys-Phe-Leu-NH2) and established criteria for selecting Ser/Thr, Xaa, and Zaa substitutions optimal for specific R-1-Ser/Thr peptide bond hydrolysis in the presence of Ni(II) ions (Krezel, A.; Kopera, E.; Protas, A. M.; Poznanski, J.; Wysouch-Cieszynska, A.; Bal, W. J. Am. Chem. Soc. 2010, 132,3355-3366). The screening results were confirmed by kinetic studies of hydrolysis of seven peptides: R-1-Ser-Arg-His-Trp-R-2, R-1-Ser-Lys-His-Trp-R-2, R-1-Ser-Ala-His-TrP-R-2, R-1-Ser-Arg-His-Ala-R-2, R-1-Ser-Gly-His-Ala-R-2, R-1-Thr-Arg-His-Trp-R-2, and R-1-Thr-His-His-Trp-R-2. In this paper, we used the same seven peptides to investigate the molecular mechanism of the hydrolysis reaction. We studied temperature dependence of the reaction rate at temperatures between 24 and 75 degrees C, measured stability constants of Ni(II) complexes with hydrolysis substrates and products, and studied the course of R-1-Ser-Arg-His-Trp-R-2 peptide hydrolysis under a broad range of conditions. We established that the specific square planar complex containing the Ni(II) ion bonded to the His imidazole nitrogen and three preceding peptide bond nitrogens (4N complex) is required for the reaction to occur. The reaction mechanism includes the N-O acyl shift, yielding an intermediate ester of RI with the Ser/Thr hydroxyl group. This ester hydrolyzes spontaneously, yielding final products. The Ni(II) ion activates the R-1-Ser peptide bond by destabilizing it directly through peptide nitrogen coordination and, indirectly, by imposing a strain in the peptide chain.