Functional Analysis of the Propeptide of Subtilisin E as an Intramolecular Chaperone for Protein Folding

Abstract

The amino-terminal propeptide, consisting of 77 amino acid residues, is known to be required as an intramolecular chaperone to guide the folding of mature subtilisin E, a serine protease, into active mature enzyme. Many mutations within the pro-sequence have been shown to abolish the production of active subtilisin E (Kobayashi, T., and Inouye, M. (1992) J. Mol. Biol. 226, 931-933). Here we report characterization, refolding, and inhibitory abilities of six single amino acid substitution mutations (Ile-67 → Val, Ile-48 → Thr, Gly-44 → Asp, Lys-36 → Glu, Ala-30 → Thr, and Pro-15 → Leu) and a nonsense mutation (N59-mer) at the codon for Lys-18. These mutant propeptides were expressed in Escherichia coli using a T7 expression system and were purified to homogeneity. Surprisingly, Lys-36 → Glu, Ala-30 → Thr and Pro-15 → Leu were found to still function as a chaperone for in vitro refolding of denatured subtilisin BPN' with 60, 80, and 54% efficiency compared to the wild-type propeptide, respectively. The K(i) values against subtilisin BPN' were 1.6 x 10-9 M, 1.2 x 10-9 M, and 2.1 x 10-9 M, respectively, almost identical to the K(i) value exhibited by the wild-type propeptide (1.4 x 10-9 M). In contrast, Ile-67 → Val and Gly-44 → Asp were able to refold denatured subtilisin BPN' with only 18 and 13% efficiencies and had K(i) values of 10 and 11 x 10-9 M, respectively. The Ile-48 → Thr mutant propeptide was unable to refold denatured subtilisin BPN' and gave a 100-fold higher K(i) (118 x 10-9 M) than the wild-type propeptide. The N59-mer propeptide extending from Leu-19 to Met-78 was unable to function as a chaperone. Like the wild-type propeptide, none of the mutant propeptides had secondary structures as judged by their circular dichroism spectra. The present results demonstrate that the ability of the propeptide as a chaperone to refold the denatured protein is well correlated with its ability as a competitive inhibitor for the active enzyme. This supports the notion that the secondary and tertiary structures of the propeptide are identical or highly homologous between the renatured propeptide-subtilisin complex and the inhibitory complex formed between the propeptide and the active enzyme.