Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries
Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries
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DOI：
10.1021/bi0014195
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发表时间：
2001-02-13
期刊：
影响因子：
2.9
通讯作者：
Egmond, MR
中科院分区：
文献类型：
--
作者：
Dekker, N;Cox, RC;Egmond, MR
大肠杆菌外膜蛋白酶T （OmpT）是一种内多肽酶，可以在两个连续的基本残基之间特异性地进行切割。在这项研究中，我们利用空间定位的SPOT肽库研究了OmpT的底物特异性。在纤维素膜上直接合成了乙酰- dap (dnp)-Ala-Arg (Abz)-Gly肽。该肽含有氨基苯甲酰（Abz）荧光基团，其内部被二硝基苯（dnp）部分猝灭。用小的水溶性蛋白酶胰蛋白酶处理斑点膜，产生高荧光肽斑点。然而，与洗涤剂溶解的OmpT（估计分子质量为180 kDa的大分子复合物）孵育后，没有观察到肽切割。这个问题可以通过在膜载体和肽之间引入一个长极性聚氧乙二醇连接剂来解决。用OmpT筛选底物中P-2、P-1、P-1′和P-2′位置的肽库，重新合成阳性位点的肽，并在溶液中进行动力学测量。最佳底物Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly的周转数k（cat）为40 s(-1)，是起始底物的12倍。在P1或P-2'处含有酸性残基的肽不是OmpT的底物，这表明远程静电相互作用对酶-底物复合物的形成很重要。OmpT在PI位点对l -氨基酸具有高度选择性，但在P-1位点的选择性较低，其中P-1位点上含有d -精氨酸的肽是竞争性抑制剂（Ki为19 muM）。基于这些发现，开发了一种亲和层析树脂，可以一步从细菌裂解物中纯化OmpT。讨论了确定的一致底物序列（Arg/Lys）向下箭头(Arg/Lys)-Ala对OmpT防御抗菌肽的生物学功能的影响。
Escherichia coli outer membrane protease T (OmpT) is an endopeptidase that specifically cleaves between two consecutive basic residues. Ln this study we have investigated the substrate specificity of OmpT using spatially addressed SPOT peptide libraries. The peptide acetyl-Dap(dnp)-Ala-Arg down arrow Arg-Ala-Lys(Abz)-Gly was synthesized directly onto cellulose membrane. The peptide contained the aminobenzoyl (Abz) fluorophore, which was internally quenched by the dinitrophenyl (dnp) moiety. Treatment of the SPOT membrane with the small, water-soluble protease trypsin resulted in highly fluorescent peptide SPOTs. However, no peptide cleavage was observed after incubation with detergent-solubilized OmpT, a macromolecular complex with an estimated molecular mass of 180 kDa. This problem could be solved by the introduction of a long, polar polyoxyethylene glycol linker between the membrane support and the peptide. Peptide libraries for the P-2, P-1, P-1', and P-2' positions in the substrate were screened with OmpT, and peptides of positive SPOTs were resynthesized and subjected to kinetic measurements in solution. The best substrate Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly had a turnover number k(cat) of 40 s(-1) which is 12-fold higher than the starting substrate. Peptides containing an acidic residue at P1 or P-2' were not substrates for OmpT, suggesting that long-range electrostatic interactions are important for the formation of the enzyme-substrate complex. OmpT was highly selective toward L-amino acids at PI but was less so at P-1' where a peptide with D-Arg at P-1' was a competitive inhibitor (Ki of 19 muM) An affinity chromatography resin based on these findings was developed, which allowed for the one-step purification of OmpT from a bacterial lysate. The implications of the determined consensus substrate sequence (Arg/ Lys)down arrow (Arg/Lys)-Ala for the proposed biological function of OmpT in defense against antimicrobial peptides are discussed.