PRO-PEPTIDE AS AN INTERMOLECULAR CHAPERONE - RENATURATION OF DENATURED SUBTILISIN-E WITH A SYNTHETIC PRO-PEPTIDE

The amino-terminal pro-sequence consisting of 77 amino acid residues is required to guide the folding of secreted subtilisin E, a serine protease, into active, mature enzyme (Ikemura et al., 1987). Furthermore, denatured subtilisin E can be folded to active enzyme in an intermolecular process with the aid of an exogenously added pro-subtilisin E, the active site of which was mutated (Zhu et al., 1989). In this report, we have synthesized the pro-peptide of 77 residues (corresponding to -1 to -77 in the sequence, where residue +1 is the N-terminal amino acid residue of the mature protein), and have found that it could intermolecularly complement the folding of denatured subtilisin E to active enzyme. Furthermore, we have found that the synthetic pro-peptide exhibits specific strong binding to the active mature enzyme by inhibiting it competitively at its active centre with an upper limit to a K(i) of 5.4 x 10(-7). In contrast, synthetic pro-peptides corresponding to -44 to -77, -1 to -64 and -1 to -43 inhibited the enzyme with K(i) values weaker by two orders of magnitude. The results indicate that the sequence extending from -1 to -77 is essential for specificity of interaction, perhaps generating a conformation that accounts for both roles found hitherto, i.e. specific binding to the active centre, and guiding of the refolding to active enzyme. Thus these results suggest that the pro-peptide functions as an intermolecular chaperone.