tailieunhanh - Báo cáo Y học: Rodent a-chymases are elastase-like proteases

Although thea-chymasesof primates anddogs areknownas chymotrypsin-like proteases, the enzymatic properties of rodent a-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (. alanine, isoleucine, valine) in the P1 site of substrates | Eur. J. Biochem. 269 5921-5930 2002 FEBS 2002 doi Rodent a-chymases are elastase-like proteases Yuichi Kunori1. Masahiro Koizumi1 Tsukio Maseai1. Hidenori Kasai1 Hiroshi Kawabata1 Yuzo Yamazaki2 and Akiyoshi Fukamizu3 1TEIJIN Institute for Biomedical Research Hino Tokyo Japan 2TEIJIN Material Analysis Research Laboratories Tokyo Japan 3Center for Tsukuba Advanced Research Alliance University of Tsukuba Tsukuba Ibaraki Japan Although the a-chymases of primates and dogs are known as chymotrypsin-like proteases the enzymatic properties of rodent a-chymases rat mast cell protease 5 rMCP-5 and mouse mast cell protease 5 mMCP-5 have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s MCP-5s have a clear preference for small aliphatic amino acids . alanine isoleucine valine in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5 and found that the mutant possessing a Gly substitution of the Val at position 216 V216G lost elastase-like activity but acquired chymase activity suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases rMCP-2 human cathepsin G and human chymase revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent a-chymases have unique biological activity different from the chymases of other species. Keywords mast cell protease s chymase elastase chymotrypsin substrate specificity site-directed mutagenesis homology modeling. Chymase is a chymotrypsin-like serine protease expressed .