Evidence that highly conserved residues of Delonix regia trypsin inhibitor are important for activity.

Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44-Cys89 and Cys139-Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4-5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139-Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44-Cys89) is not required.