Site-directed mutagenesis evidence for a negatively charged trypsin inhibitory loop in sweet potato sporamin.

Sporamin, a sweet potato tuberous storage protein, has trypsin inhibitory activity. Sequence comparison with other plant trypsin inhibitors (TIs) of the Kunitz family reveals that, instead of the conserved Arg or Lys found in other Kunitz TIs, sporamin contains a negatively charged residue (Asp70 or Glu72) at the P1 reactive site. Using site-directed mutagenesis, six mutants were generated containing substitutions at the reactive site and at one of the disulfide bonds, and the recombinant proteins were assayed for TI activity. Mutants Asp70Val and Glu72Arg were found to have only 2-3% of the wild-type activity. These results provide the first evidence for a negatively charged trypsin inhibitory loop and a new mechanism of trypsin inhibition in the Kunitz family.