Porin bearing the protease activity : its structure and function

我们发现铜绿假单胞菌外膜上的蛋白D2（OprD）孔蛋白具有蛋白酶活性。这是从以下结果得出的结论。(a)高度纯化的OprD样品根据Michaelis-Menten动力学水解合成肽，（B），二异丙基氟磷酸（DFP），一种丝氨酸蛋白酶特异性抑制剂，抑制水解活性并与OprD蛋白共价结合，和（c），针对OprD蛋白产生的单克隆抗体以浓度依赖性方式抑制蛋白酶活性。然而，人们可能怀疑蛋白酶活性可能由OprD制剂中非常少量的蛋白酶介导。因此，为了最终证明OprD蛋白中的蛋白酶活性并准确鉴定OprD蛋白酶的催化残基，我们构建了用谷氨酰胺、天冬酰胺或丙氨酸取代His 156、Asp 208或Ser 296的突变OprD蛋白，因为根据OprD和OprD之间的氨基酸序列的比较分析，预测这些氨基酸是催化三联体组分，丝氨酸蛋白酶如胰蛋白酶。将这些菌株的外膜中表达的野生型和突变体OprD蛋白纯化至均一，并进行蛋白酶测定。因此，所有三种突变体显示具有小于野生型OprD活性的0.1%的蛋白酶活性。该结果最终证明OprD蛋白自身具有蛋白酶，并且His 156、Asp 208和Ser 296残基是OprD蛋白酶的催化三联体的组成部分。此外，我们研究了这些催化残基与亚胺培南，碳青霉烯类之一的相互作用的作用，因为OprD被证明有亚胺培南的结合位点。这些突变株对imipem的敏感性表明，His 156和Asp 208可能在imipem通过OprD蛋白的快速扩散中起一定作用，而Ser 296可能不起作用。

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.