Porin bearing the protease activity : its structure and function

我们发现铜绿假单胞菌外膜的蛋白D2 （OprD）孔蛋白具有蛋白酶活性。这是由以下结果得出的结论。(a)高纯度的OprD样品根据Michaelis-Menten动力学水解合成肽，(b)丝氨酸蛋白酶特异性抑制剂氟磷酸二异丙酯（DFP）抑制水解活性并与OprD蛋白共价结合，(c)针对OprD蛋白的单克隆抗体以浓度依赖的方式抑制蛋白酶活性。然而，人们可能会怀疑，在OprD制剂中，蛋白酶活性可能是由非常少量的蛋白酶介导的。因此，为了最终证明OprD蛋白的蛋白酶活性，同时鉴定OprD蛋白酶的催化残基，我们构建了突变体OprD蛋白，分别用谷氨酰胺、天冬酰胺或丙氨酸取代His156、Asp208或Ser296，因为从OprD与丝氨酸蛋白酶（如胰蛋白酶）的氨基酸序列比较分析中预测这些氨基酸是催化三联体成分。在这些菌株的外膜上表达的野生型和突变型OprD蛋白被纯化并进行了蛋白酶试验。结果显示，所有三个突变体的蛋白酶活性都低于野生型OprD活性的0.1%。这一结果表明OprD蛋白本身具有该蛋白酶，并且His156、Asp208和Ser296残基是OprD蛋白酶催化三联体的组成部分。此外，我们研究了这些催化残基对亚胺培南相互作用的作用，亚胺培南是碳青霉烯类之一，因为OprD被证明具有亚胺培南的结合位点。这些突变株对imipem的易感性表明，His156和Asp208而非Ser296可能在imipem通过OprD蛋白的加速扩散中起一定作用。

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.