Inactivation of Acacia confusa trypsin inhibitor by site‐specific mutagenesis

FEBS Letters

Lin, Jung-Yaw¹  Lee, Ming-Chih¹  Hung, Chih-Hung¹ 

[1] Institute of Biochemistry, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Rd, Taipei, Taiwan 10018, ROC

关键词: Kunitz type trypsin inhibitor;    Disulphide bond;    Inhibitor active site;    ACTI;    Acacia confusa trypsin inhibitor;    L-BAPNA;    N α-benzoyl-l-arginine-4-nitro-anilide hydrochloride;    BTEE;    N-benzoyl-l-tyrosine ethyl ester;    re;    recombinant;    GST;    glutathione-S-transferase;    SDS-PAGE;    sodium dodecylsulfate-polyacrylamide gel electrophoresis;    M r;    relative molecular mass;   

DOI  :  10.1016/0014-5793(94)01066-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Native Acacia confusa trypsin inhibitor (ACTI) contains two disulphide bonds; one is an intrachain disulphide bond (Cys⁴⁰-Cys⁸⁶), located in the A-chain, while the other is an interchain disulphide bond (Cys¹³³-Cys¹⁴¹) connecting the A- and B-chain; the inhibitor consists of 175 amino acid residues, 136 residues in the A-chain and 39 residues in the B-chain. The putative reactive site of ACTI is located at Lys⁶⁴, while for all other Kunitz family trypsin inhibitors it is at Arg⁶⁴. When the Lys⁶⁴ residue of ACTI was converted into Ile or Arg by site-specific mutagenesis, the K64I mutant completely lost its inhibitory activity but the K64R mutant retained most of its inhibitory activity. The C133G mutant lost its inhibitory activity while the C40G mutant did not. This suggests that the interchain disulphide bond (Cys¹³³-Cys¹⁴¹) linking two β-strands of the six-strand β-barrel is essential for ACTI inhibitory activity, while the intrachain disulphide bond (Cys⁴⁰-Cys⁸⁶) connecting the two loops is non-essential.

【 授权许可】

Unknown