Cleavage specificity of the subtilisin-like protease C1 from soybean

The cleavage specificity of protease C1, isolated from soybean (Glycine max (L.) Merrill) seedling cotyledons, was examined using oligopeptide substrates in an HPLC based assay. A series of peptides based on the sequence Ac-KVEKEESEEGE-NH₂ was used, mimicking a natural cleavage site of protease C1 in the α subunit of the storage protein β-conglycinin. A study of substrate peptides truncated from either the N- or C-terminus indicates that the minimal requirements for cleavage by protease C2 are three residues N-terminal to the cleaved bond, and two residues C-terminal (i.e. P₃-P₂′). The maximal rate of cleavage is reached with substrates containing four to five residues N-terminal to the cleaved bond and four residues C-terminal (i.e. P₄ or P₅ to P₄′). The importance of Glu residues at the P₁, P₁′, and P₄ positions was examined using a series of substituted nonapeptides (P₅-P₄′) with a base sequence of Ac-KVEKEESEE-NH₂. At the P₁ position, the relative ranking, based on k_cat/K_m, was E>Q>K>A>D>F>S. Substitutions at the P₁′ position yield the ranking E≅Q>A>S>D>K>F, while those at P₄′ had less effect on k_cat/K_m, yielding the ranking F≅S≅E≅D>K>A≅Q. These data show that protease C1 prefers to cleave at Glu-Glu and Glu-Gln bonds, and that the nature of the P₄′ position is less important. The fact that there is specificity in the cleavage of the oligopeptides suggests that the more limited specific cleavage of the α and α′ subunits of β-conglycinin by protease C1 is due to a combination of the sequence cleavage specificity of the protease and the accessibility of appropriate scissile peptide bonds on the surface of the substrate protein.