Functional analysis of the propeptide of subtilisin E as an intramolecular chaperone for protein folding. Refolding and inhibitory abilities of propeptide mutants.
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 Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively. The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively, almost identical to the Ki 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 and13% efficiencies and had Ki 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 Ki (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.
Li
Y
Y
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry-New Jersey, Piscataway 08854, USA.
Hu
Z
Z
Jordan
F
F
Inouye
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004792:Enzyme Precursors / Q000502:physiology*; D018832:Molecular Chaperones / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry*
D004792:Enzyme Precursors; D018832:Molecular Chaperones; D013381:Subtilisins