Inactivation of Acacia confusa trypsin inhibitor by site-specific mutagenesis.
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 beta-strands of the six-strand beta-barrel is essential for ACTI inhibitory activity, while the intrachain disulphide bond (Cys40-Cys86) connecting the two loops is non-essential.
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, ROC.
C H
CH
Hung
M C
MC
Lee
J Y
JY
Lin
D000045:Acacia; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008239:Lysine / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014361:Trypsin Inhibitors / Q000235:genetics* / Q000378:metabolism*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; C074637:ACTI protein, Acacia confusa; D008239:Lysine