| Budget Amount *help |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1991: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Research Abstract |
Achromobacter protease I (API) is an extracellular protease isolated from Achromobacter lyticus and is specific to lysyl bonds including Lys-Pro bond. We have investigated the structural basis of the specificity for lysine and tried to alter the specificity of API by protein engineering technique.
Two acidic amino acids, Glu190 and Asp225, of API have been found as candidates for the specificity determinant based on amino acid sequence comparison. To determine which residue is responsible for lysine-specificity, we have undertaken to prepare several mutants at Glu190 ro Asp225. Glu190 was replaced by Asp, Gln or Leu and all mutants were secreted to the periplasm as a mature protein. These three mutants were fully active and bore the same substrate specificity as native API. Asp225 was substituted for Glu, Asp or Leu. A mutant replaced by Glu was purified as a mature active protein, but the activity was substantially diminished. The two other mutants were detected as inactive precursor p
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roteins in the periplasm. Immaturation of the precursor indicated a loss of the catalytic activity in the mutants. These results suggest that Asp225 is essential for the hydrolytic activity of API for lysyl bonds.
Recently, the three dimensional structure of API has been elucidated by X-ray crystallography. The tertiary fold and the structure of the active site are very similar between API and bovine trypsin. The critical structural difference in the S1 substrate binding subsite is the shallow binding pocket. The Asp225 is located at the wall of the pocket. Its side chain sticks out inside the pocket, making the bottom of the pocket 2 A* shallower than that of trypsin. In API, the side chain of lysine of the substrate can fit in the pocket and bind to Asp225 by electrostatic interaction. Since the side chain of arginine is longer than that of lysine, arginine has difficulty fitting into the shallow binding pocket of API. In order to alter the specificity of API to arginine, Asp225 and Vall88 were replaced by Glu and Asp, respectively, making the pocket deeper. However, this mutant was not expressed in E. coli either as precursor or mature protein. The reason why the mutant is not expressed is unclear at present. Less
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