Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P(1) and P(1)' residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor.
Two peptides, SGCI and SGTI, that inhibited chymotrypsin and trypsin, respectively, were isolated from the haemolymph of Schistocerca gregaria. Their primary structures were found to be identical with SGP-2 and SGP-1, two of a series of peptides isolated from ovaries of the same species (A. Hamdaoui et al., FEBS Lett. 422 (1998) 74-78). All these peptides are composed of 35-36 amino acid residues and contain three homologous disulfide bridges. The residues imparting specificity to SGCI and SGTI were identified as Leu-30 and Arg-29, respectively. The peptides were synthesised by solid-phase peptide synthesis, and the synthetic ones displayed the same inhibition as the natural forms: SGCI is a strong inhibitor of chymotrypsin (K(i) = 6.2 x 10(-12) M), and SGTI is a rather weak inhibitor of trypsin (K(i) = 2.1 x 10(-7) M). The replacement of P(1) then P(1)' residues of SGCI with trypsin-specific residues increased affinity towards trypsin 3600- and 1100-fold, respectively, thus SGCI was converted to a strong trypsin inhibitor (K(i) = 5.0 x 10(-12) M) that retained some inhibitory affinity towards chymotrypsin (K(i) = 3.5 x 10(-8) M). The documented role of both P(1) and P(1)' highlights the importance of S(1)'P(1)' interactions in enzyme-inhibitor complexes.