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 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.
Highlights
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
PCR random mutagenesis, a total of 25 single amino acid substitution mutations were isolated that affected the production of active subtilisin in vivo (Kobayashi and Inouye, 1992)
The Ki values were found to be well correlated with the refolding abilities of the class I mutant propeptides; AlaϪ30 3 Thr showed 80% refolding efficiency with Ki of 1.15 ϫ 10Ϫ9 M, LysϪ36 3 Glu 60% with Ki of 1.61 ϫ 10Ϫ9 M, and ProϪ15 3 Leu 54% with Ki of 2.10 ϫ 10Ϫ9 M
Summary
From the ‡Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry-New Jersey, Piscataway, New Jersey 08854 and the §Department of Chemistry, Rutgers, State of University of New Jersey, Newark, New Jersey 07102. We report characterization, refolding, and inhibitory abilities of six single amino acid substitution mutations (Ile؊67 3 Val, Ile؊48 3 Thr, Gly؊44 3 Asp, Lys؊36 3 Glu, Ala؊30 3 Thr, and Pro؊15 3 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. A number of mutations within the propeptide have been isolated, which are defective in producing active subtilisin, indicating that specific amino acid residues and/or regions in the pro-sequence play important roles in the process of folding the mature subtilisin (Kobayashi and Inouye, 1992). We selected six of the 25 mutations previously isolated, and overexpressed them using a T7 expression system in Escherichia coli These mutant propeptides were purified to near homogeneity, and their abilities to refold denatured subtilisin in vitro were examined. The efficiencies of the mutant propeptides were found to correlate well to the Ki values exhibited by these propeptides,
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