Inactivation of Acacia Confusa Trypsin Inhibitor by Site-Specific Mutagenesis

10.30047/JGMB.199412.0009

Chih-Hungg Hung；Ming-Chih Lee；Jung-Yaw Lin

Journal of Genetics and Molecular Biology

5卷4期（1994 / 12 / 01）

78 - 78

英文

ACTI is comprised of two chains, namely A and B chain linked by the disulphide bond between Cys(133) and Cys(141), and the inhibitor consists of 175 amino acid residues, 136 residues in the A chain and 39 residues the B chain. The putative reactive site of ACTI is located at Lys64 residue while all other Kunitz family trypsin inhibitors, at Arg64 residue. When the Lys64 residue of ACTI was converted into Ile or Arg residue by site-specific mutagenesis, the mutant (K64I) completely lost its inhibitory activity but the mutant (K64R) retained most of its inhibitory activity. Native ACTI contains two disulphide bonds; one is an intra-disulphide bond (CyS40-CyS86), located in A-chain while the other is an inter-disulphide bond (Cys133-cys141), in A and B-chain. The reACTI of single polypeptide chain without post-translation processing by proteoloysis is biologically active. The mutant (C133G) lost its inhibitory activity while the mutant (C40G) did not. It suggests that the inter-disulphide bond (Cys133-Cys141) linking between two b-strands of six-strand b barrel is indepensible for ACTI inhibitory activity while the intradisulphide bond (Cys40-Cys86) connecting two loops is dispensible based on the 3-D structure of Erythrina caffra trypsin inhibitor which amino acid sequence has a high degree of homology to that of ACTI.