Pro‐peptide as an intermolecular chaperone: renaturation of denatured subtilisin E with a synthetic pro‐peptide

Abstract

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 Ki 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 Ki 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 intramolecular chaperone [corrected].

Folding pathway mediated by an intramolecular chaperone.

“…Under certain conditions, however, it is possible for an intermolecular cleavage to occur. In both cases the propeptide acts as a substrate for subtilisin (7). Moreover, the cleavage is mediated by the same catalytic triad-namely, Asp32, His64, and Ser221.…”

“…Earlier work had shown the inability of Gdn-HCldenatured subtilisin to refold by itself to a biologically active state (6)(7)(8)15 (17).…”

“…Using subtilisin E as a model system, work in our laboratory has unambiguously shown that the 77-amino acid propeptide plays a vital role in folding the 275 amino acids of the mature enzyme (6). Because the propeptide is covalently attached to the N terminus of the mature enzyme prior to its maturation, and because each propeptide molecule mediates the folding of one enzyme molecule (generally the corresponding molecule), the term intramolecular chaperones has been coined for such propeptides to distinguish them from chaperones (6)(7)(8). Concurrent with this proposition, it was shown that the 13-amino acid propeptide of bovine pancreatic trypsin inhibitor facilitated the in vitro folding pathway by acting as a tethered, solvent-accessible, intramolecular thiol-disulfide reagent (9).…”

Sorting and processing of secretory proteins

“…By expressing active furin together with various mutant furins, these authors confirm that activation occurs by an intramolecular autoproteolytic mechanism. Whether the furin pro-region is important for the proper folding of the enzyme, as is the case for subtilisin E [259][260][261][262], has not yet been determined. Further processing at as yet unidentified sites immediately Nterminal to the trans-membrane domain is also known to occur, leading to the generation of soluble (and released) 76-80 kDa forms [222,263].…”

Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

“…Whereas the activation of some serine proteinases that have short proregions, like trypsin and chymotrypsin, is well known, it is less clear for enzymes with longer proregions ( > 60 residues). The proregions of these proteinases are essential for such physiological functions as intracellular trafficking, correct folding of the enzyme during maturation, and inhibition of the mature enzyme [1][2][3][4][5]. The inhibition of proteinases by their respective proregions is a rather specific process [6,7], and this relationship can be used to develop a new generation of specific peptide inhibitors.…”