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 a ctivity, Sequence comparison with other plant trypsin inhibitors (TIs) of t he Kunitz family. reveals that, instead of the conserved Arg or Lys found i n other Kunitz TIs, sporamin contains a negatively charged residue (Asp70 o r Glu72) at the P1 reactive site. Using site-directed mutagenesis, six muta nts were generated containing substitutions at the reactive site and at one of the disulfide bonds, and the recombinant proteins were assayed for TI a ctivity. Mutants Asp70Val and Glu72Arg were found to have only 2-3% of the wild-type activity, These results provide the first evidence for a negative ly charged trypsin inhibitory loop and a new mechanism of trypsin inhibitio n in the Kunitz family. (C) 2001 Published by Elsevier Science B.V. on beha lf of the Federation of European Biochemical Societies.