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 alpha subunit of the storage protein beta-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- P-3-P-2'). 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. P-4 or P-5 to P-4'). The importance of Glu residues at the P-1, P-1', and P-4 positions was examined using a series of substituted nonapeptides (P-5-P-4') with a base sequence of Ac-KVEKEESEE-NH2 At the P-1 position, the relative ranking, based on k(cat)/K-m, was E > Q > K > A > D > F > S. Substitutions at the P, position yield the ranking E congruent to Q > A > S > D > K > F, while those at P-4' had less effect on k(cat)/K-m, yielding the ranking F congruent to S congruent to E congruent to D > K > A congruent to Q. These data show that protease C I prefers to cleave at Glu-Glu and Glu-Gln bonds, and that the nature of the P-4' 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 alpha and alpha' subunits of beta-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. (C) 2002 Elsevier Science B.V. All rights reserved.