Pro-peptide as an intramolecular chaperone: renaturation of denatured subtilisin E with a synthetic pro-peptide [corrected].
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].
Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854-5635.
Y
Y
Ohta
H
H
Hojo
S
S
Aimoto
T
T
Kobayashi
X
X
Zhu
F
F
Jordan
M
M
Inouye
D018833:Chaperonins; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011506:Proteins; C057107:prosubtilisin; D013381:Subtilisins; D018833:Chaperonins