Cleavage specificity of the subtilisin-like protease C1 from soybean

Abstract

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.