Laboratory of Protein Engineering, Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland.
J Am Chem Soc. 2010 Mar 17;132(10):3355-66. doi: 10.1021/ja907567r.
Previously we demonstrated for several examples that peptides having a general internal sequence R(N)-Yaa-Ser/Thr-Xaa-His-Zaa-R(C) (Yaa = Glu or Ala, Xaa = Ala or His, Zaa = Lys, R(N) and R(C) = any N- and C-terminal amino acid sequence) were hydrolyzed specifically at the Yaa-Ser/Thr peptide bond in the presence of Ni(II) ions at alkaline pH (Krezel, A., Mylonas, M., Kopera, E. and Bal, E. Acta Biochim. Polon. 2006, 53, 721-727 and references therein). Hereby we report the synthesis of a combinatorial library of CH(3)CO-Gly-Ala-(Ser/Thr)-Xaa-His-Zaa-Lys-Phe-Leu-NH(2) peptides, where Xaa residues included 17 common alpha-amino acids (except Asp, Glu, and Cys) and Zaa residues included 19 common alpha-amino acids (except Cys). The Ni(II)-dependent hydrolysis at 37 and 45 degrees C of batches of combinatorial peptide mixtures randomized at Zaa was monitored by MALDI-TOF mass spectrometry. The correctness of library-based predictions was confirmed by accurate measurements of hydrolysis rates of seven selected peptides using HPLC. The hydrolysis was strictly limited to the Ala-Ser/Thr bond in all library and individual peptide experiments. The effects of individual residues on hydrolysis rates were quantified and correlated with physical properties of their side chains according to a model of independent contributions of Xaa and Zaa residues. The principal component analysis calculations demonstrated partial molar side chain volume and the free energy of amino acid vaporization for both Xaa and Zaa residues and the amine pK(a) for Zaa residues to be the most significant empirical parameters influencing the hydrolysis rate. Therefore, efficient hydrolysis required bulky and hydrophobic residues at both variable positions Xaa and Zaa, which contributed independently to the hydrolysis rate. This relationship between the peptide sequence and the hydrolysis rate provides a basis for further research, aimed at the elucidation of the reaction mechanism and biotechnological applications of Ni(II)-dependent peptide bond hydrolysis.
先前，我们已经用几个实例证明了具有一般内部序列 R（N）-Yaa-Ser/Thr-Xaa-His-Zaa-R（C）的肽（Yaa = Glu 或 Ala，Xaa = Ala 或 His，Zaa = Lys，R（N）和 R（C）= 任何 N-和 C-末端氨基酸序列）在碱性 pH 下存在 Ni（II）离子时可以特异性地在 Yaa-Ser/Thr 肽键处水解（Krezel，A.，Mylonas，M.，Kopera，E. 和 Bal，E. Acta Biochim. Polon. 2006，53，721-727 和其中的参考文献）。在此，我们报告了 CH（3）CO-Gly-Ala-（Ser/Thr）-Xaa-His-Zaa-Lys-Phe-Leu-NH（2）肽的组合文库的合成，其中 Xaa 残基包括 17 种常见的α-氨基酸（除 Asp、Glu 和 Cys 外），Zaa 残基包括 19 种常见的α-氨基酸（除 Cys 外）。通过 MALDI-TOF 质谱监测 37 和 45°C 下分批组合肽混合物在 Zaa 处的 Ni（II）依赖性水解，使用 HPLC 准确测量了七种选定肽的水解速率，从而验证了文库预测的正确性。结果表明，在所有文库和个别肽实验中，水解均严格限于 Ala-Ser/Thr 键。定量研究了单个残基对水解速率的影响，并根据 Xaa 和 Zaa 残基独立贡献的模型与它们侧链的物理性质相关联。主成分分析计算表明，对于 Xaa 和 Zaa 残基，摩尔侧链体积和氨基酸蒸发自由能以及 Zaa 残基的胺 pK（a）是影响水解速率的最显著经验参数。因此，有效的水解需要在两个可变位置 Xaa 和 Zaa 处都具有大体积和疏水性残基，它们独立地促进水解速率。这种肽序列与水解速率之间的关系为进一步的研究提供了基础，旨在阐明 Ni（II）依赖性肽键水解的反应机制和生物技术应用。
