Substrate specificity of cucumisin on synthetic peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4 , n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Afa-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the i ncrease of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-A la-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentia lly same with the increase of n = 2 to 3. Then, it was concluded that cucum isin has a SS-S; subsite length. In order to identify the substrate specifi city at P-1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pr o, Asp, Glu, Lys, Arg, Asn, Gin, Phe, Tyr, Ser, Thr, Met, Trp, His) were sy nthesized and digested by cucumisin. Cucumisin showed broad specificity at the P-1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. M oreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did n ot cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys e specially Ser. The specificity of cucumisin for naturally occurring peptide s does not agree strictly with the specificity obtained by synthetic peptid es at the P-1 or P-1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain su itable amino acid residues at P-1 and (or) P-1' positions. Moreover, cucumi sin prefers Pro than Leu at P-2 position, indicating that the specificity a t P-2 position differs from that of papain.