Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca²⁺. PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, and , were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases.

中文翻译：

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使用合成肽对噬菌体Pseudomurein内肽酶PeiW和PeiP进行生化表征。

假单脲尿素内肽酶通过裂解假单脲尿素的肽链中的异肽键Ala- ε- Lys引起产甲烷菌的细胞壁溶解。PeiW和PeiP是由狼毒甲烷杆菌的噬菌体ΨM100和马氏甲烷球菌属的噬菌体ΨM1和ΨM2编码的两种热稳定的假粘菌素内切肽酶， 分别。开发了使用合成肽底物的连续测定法，并将其用于重组PeiW和PeiP的生化表征。这些合成肽底物优于天然底物的优势是灵敏度，高纯度和表征，以及它们比天然底物更容易获得的事实。在还原剂的存在下，纯化的PeiW和PeiP在有氧和厌氧条件下均显示出相似的活性。两种酶都需要二价金属才能发挥活性，并且在存在Ca ²⁺的情况下表现出更高的热稳定性。PeiW和PeiP在催化中涉及半胱氨酸残基，并具有单体天然构象。确定了动力学参数和，以及ε丙氨酸和赖氨酸之间的β-异肽键被确认为伪尿素中这些酶裂解的键。这种新的测定方法可能对肽酶的一般研究以及鉴定易受特定假粘蛋白内异肽酶裂解的特定产甲烷菌有更广泛的应用。