Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany.
Department of Bioinorganic Chemistry, Institute of Chemistry, University of Hohenheim, Stuttgart, Germany.
Enzyme Microb Technol. 2018 Mar;110:69-78. doi: 10.1016/j.enzmictec.2017.10.002. Epub 2017 Oct 12.
The aminopeptidase A (PepA; EC 3.4.11.7) belongs to the group of metallopeptidases with two bound metal ions per subunit (M1M2(PepA)) and is specific for the cleavage of N-terminal glutamic (Glu) and aspartic acid (Asp) and, in low amounts, serine (Ser) residues. Our group recently characterized the first PepA from a Lactobacillus strain. However, the characterization was performed using synthetic para-nitroaniline substrates and not original peptide substrates, as was done in the current study. Prior to the characterization using original peptide substrates, the PepA purified was converted to its inactive apo-form and eight different metal ions were tested to restore its activity. It was found that five of the metal ions were able to reactivate apo-PepA: Co, Cu, Mn, Ni and Zn. Interestingly, depending on the metal ion used for reactivation, the activity and the pH and temperature profile differed. Exemplarily, MnMn(PepA), NiNi(PepA) and ZnZn(PepA) had an activity optimum using MES buffer (50mM, pH 6.0) and 60°C, whereas the activity optimum changed to Na/K-phosphate-buffer (50mM, pH 7.0) and 55°C for CuCu(PepA). However, more important than the changes in optimum pH and temperature, the kinetic properties of PepA were affected by the metal ion used. While all PepA variants could release N-terminal Glu or Asp, only CoCo(PepA), NiNi(PepA) and CuCu(PepA) could release Ser from the particular peptide substrate. In addition, it was found that the enzyme efficiency (V/K) and catalytic mechanism (positive cooperative binding (Hill coefficent; n), substrate inhibition (K)) were influenced by the metal ion. Exemplarily, a high cooperativity (n>2),K value >20mM and preference for N-terminal Glu were detected for CuCu(PepA). In summary, the results suggested that an exchange of the metal ion can be used for tailoring the properties of PepA for specific hydrolysis requirements.
氨肽酶 A（PepA；EC 3.4.11.7）属于金属肽酶组，每个亚基含有两个结合的金属离子（M1M2(PepA)），特异性切割 N 端的谷氨酸（Glu）和天冬氨酸（Asp），并且在低浓度下也可切割丝氨酸（Ser）残基。我们的研究小组最近从乳杆菌菌株中鉴定出了第一个 PepA。然而，与当前研究中使用原始肽底物不同，之前的鉴定是使用合成的对硝基苯胺底物进行的。在使用原始肽底物进行鉴定之前，将纯化的 PepA 转化为无活性的脱辅基形式，并测试了八种不同的金属离子以恢复其活性。结果发现，有五种金属离子能够使脱辅基 PepA 重新激活：Co、Cu、Mn、Ni 和 Zn。有趣的是，根据用于重新激活的金属离子的不同，活性以及 pH 和温度特性也不同。例如，MnMn(PepA)、NiNi(PepA) 和 ZnZn(PepA) 在使用 MES 缓冲液（50mM，pH 6.0）和 60°C 时具有活性最佳值，而对于 CuCu(PepA)，活性最佳值变为 Na/K-磷酸盐缓冲液（50mM，pH 7.0）和 55°C。然而，比最佳 pH 和温度的变化更重要的是，金属离子的使用会影响 PepA 的动力学特性。虽然所有 PepA 变体都可以从 N 端释放 Glu 或 Asp，但只有 CoCo(PepA)、NiNi(PepA) 和 CuCu(PepA) 可以从特定的肽底物中释放 Ser。此外，还发现酶效率（V/K）和催化机制（正协同结合（Hill 系数；n）、底物抑制（K））受金属离子的影响。例如，对于 CuCu(PepA)，检测到高协同性（n>2）、K 值>20mM 和对 N 端 Glu 的偏好。总之，研究结果表明，金属离子的交换可用于针对特定水解要求来调整 PepA 的特性。
