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Pro‐peptide as an intermolecular chaperone: renaturation of denatured subtilisin E with a synthetic pro‐peptide
Author(s) -
Ohta Y.,
Hojo H.,
Aimoto S.,
Kobayashi T.,
Zhu X.,
Jordan F.,
Inouye M.
Publication year - 1991
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1991.tb00797.x
Subject(s) - subtilisin , active site , peptide , biochemistry , chaperone (clinical) , enzyme , biology , serine protease , peptide sequence , residue (chemistry) , amino acid , protease , protein folding , proteases , stereochemistry , chemistry , medicine , pathology , gene
Summary The ammo‐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 × 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.