| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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.
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