Cleavage specificity of cucumisin, a serine protease, with synthetic substrates

The substrate specificity of a plant serine protease, cucumisin (EC 3.4.21. 25), was studied by the use of synthetic oligopeptides and peptidyl-pNA sub strates. Since P1'-Ser, Ala, and Gly substrates were hydrolyzed rapidly, cu cumisin appears to prefer a small side chain at the P1' position of the oli gopeptide substrate. The k(cat)/K-m for the hydrolysis of P1-Leu, Ala, Phe, and Glu substrates demonstrated that they were preferentially cleaved over P1-Lys, diaminopropionic acid (Dap), Gly, Val, and Pro substrates. From th e digestion of peptidyl-pNAs, the specificity of the protease was determine d to be broad, but the preferential cleavage sites were hydrophobic amino a cid residues at the P1 position. (C) 2000 Elsevier Science Ltd. All rights reserved.