The role of the P 2′ position of Bowman-Birk proteinase inhibitor in the inhibition of trypsin : Studies on P 2′ variation in cyclic peptides encompassing the reactive site loop

Abstract

The role of the P 2′ residue in proteinase inhibitors of the Bowman-Birk family was investigated using synthetic cyclic peptides based on the reactive site loop of the inhibitor. A series of 21 variants having different P 2′ residues was tested for inhibition of trypsin, and the rate at which they were hydrolysed by this enzyme was also measured. Variation at P 2′ was found to result in marked differences in inhibitory potency, with the best sequence (Ile) having a K i value of 9 nM. Peptides with P 2′ Gly, Pro or Glu failed to demonstrate any measurable inhibition ( K i>1 mM). The peptides also displayed significant differences in the rates at which they were hydrolysed, which varied by over three orders of magnitude between the difference sequences. There was found to be overall correlation between the K i value and the rate of hydrolysis, with peptides that inhibited best also being hydrolysed more slowly. The results are discussed in light of the sequence information for Bowman-Birk inhibitor proteins.