Porin bearing the protease activity : its structure and function

具有蛋白酶活性的孔蛋白：其结构和功能

• 批准号: 07670327
• 负责人: YOSHIHARA Eisaku
• 金额: $ 1.6万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670327/
• 关键词: Pseudomonas aeruginosa; outer membrane protein; porin; protein D2; protease; site-directed mutagenesis; catalytic residue; mutifunctional protein; protein D2; 外膜; ptoteinD2; チャンネル蛋白; 塩基性アミノ酸; モノクローナル抗体; Pseudomonas aeruginosa; outer membrane protein; porin; protein D2; protease; site-directed mutagenesis; catalytic residue; mutifunctional protein; protein D2; 外膜; ptoteinD2; チャンネル蛋白; 塩基性アミノ酸; モノクローナル抗体

We found out that protein D2 (OprD) porin in the outer membrane of Pseudomonas aeruginosa bears the protease activity. This was concluded from the following results. (a) Highly purified OprD sample hydrolyzed the synthetic peptides according to the Michaelis-Menten kinetics, (b), diisopropyl fluorophosphate (DFP), an inhibitor specific for serine protease, inhibited the hydrolytic activity and bound covalently with OprD protein, and (c), monoclonal antibody raised against OprD protein inhibited the protease activity in a concentration-dependent manner. However, one may suspect that the protease activity may be mediated by very small amounts of protease (s) in the OprD preparation. Therefore, in order to demonstrate conclusively the protease activity in OprD protein and simultaniouly identfy the catalytic residues of OprD protease, we constructed the mutant OprD proteins with the substitution of His156, Asp208 or Ser296 with glutamine, asparagine or alanine, respectively since these amino acids were predicted to be catalytic triad constituents from the comparison analysisi of the amino acid sequence between OprD and serine protease such as trypsin. The wild-type and mutant OprD proteins expressed in the outer membrane of these strains were purified to be homogeneity and were subjected to the protease assay. Consequently all the three mutants were shown to have the protease activity with less than 0.1% of the wild-type OprD activity. This result demonstrates conclusively that OprD protein itselu has the protease and that His156, Asp208 and Ser296 residues are the constituents of the catalytic triad of OprD protease. Furthermore, we studied the role of these catalytic residues to the interaction with imipenem, one of carbapenems since the OprD was shown to have the binding site to imipenem. Susceptiblities of these mutant strains agains imipem showed that His156 and Asp208 but not Ser296 may have some role for the fasilitaed diffusion of imipem through OprD protein.

我们发现，铜绿假单胞菌外膜中的蛋白D2（OPRD）旋转具有蛋白酶活性。从以下结果得出结论。 (a) Highly purified OprD sample hydrolyzed the synthetic peptides according to the Michaelis-Menten kinetics, (b), diisopropyl fluorophosphate (DFP), an inhibitor specific for serine protease, inhibited the hydrolytic activity and bound covalently with OprD protein, and (c), monoclonal antibody raised against OprD protein inhibited the protease activity in a浓度依赖性方式。但是，可能会怀疑蛋白酶活性可能是由OPRD制剂中少量蛋白酶介导的。因此，为了结论性地证明OPRD​​蛋白和同时识别OPRD蛋白酶的催化残基的蛋白酶活性，我们用His156，Asp208或Ser296代替了这些突变的OPRD蛋白，用谷氨酰胺，天素酸是育种，因为它们是抗分裂性的，因为它们是在谷氨酰胺，氨基酸方面的分析。 OPRD和丝氨酸蛋白酶（如胰蛋白酶）之间的氨基酸序列的。在这些菌株的外膜中表达的野生型和突变型OPRD蛋白被纯化为均匀性，并经过蛋白酶测定。因此，所有这三个突变体均显示出蛋白酶活性，而野生型OPRD活性的0.1％。该结果表明，OPRD蛋白Itselu具有蛋白酶，HIS156，ASP208和SER296残基是OPRD蛋白酶催化三合会的组成部分。此外，我们研究了这些催化残基在与咪毕烯的相互作用中的作用，这是碳青霉烯之一，因为OPRD被证明具有与丙咪苯成具有结合位点。这些突变菌株的敏感性再次表明，His156和Asp208而不是Ser296可能对Imipem通过OPRD蛋白的Fasilita扩散起作用。