FUNCTIONAL-ANALYSIS OF THE PROPEPTIDE OF SUBTILISIN-E AS AN INTRAMOLECULAR CHAPERONE FOR PROTEIN-FOLDING - REFOLDING AND INHIBITORY ABILITIES OF PROPEPTIDE MUTANTS
FUNCTIONAL-ANALYSIS OF THE PROPEPTIDE OF SUBTILISIN-E AS AN INTRAMOLECULAR CHAPERONE FOR PROTEIN-FOLDING - REFOLDING AND INHIBITORY ABILITIES OF PROPEPTIDE MUTANTS
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DOI：
10.1074/jbc.270.42.25127
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发表时间：
1995-10-20
影响因子：
4.8
通讯作者：
INOUYE, M
中科院分区：
文献类型：
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作者：
LI, YY;HU, ZX;INOUYE, M
已知由77个氨基酸残基组成的氨基末端前肽作为分子内伴侣来引导成熟枯草杆菌蛋白酶E（丝氨酸蛋白酶）折叠成活性成熟酶。原序列内的许多突变已显示消除活性枯草杆菌蛋白酶E的产生（小林，T.，和Inouye，M.等人（1992）J. Mol. Biol. 226，931-933）。在这里，我们报告了六个单一氨基酸取代突变（Ile（-67）-->瓦尔，Ile（-48）--> Thr，Gly（-44）-->）Asp，Lys（-36）--> Glu，Ala（-30）--> Thr和Pro（-15）--> Leu）和Lys（-18）密码子处的无义突变（N59-mer）的表征、重折叠和抑制能力。使用T7表达系统在大肠杆菌中表达这些突变体前肽，并纯化至均一。令人惊讶的是，发现Lys（-36）-> Glu、Ala（-30）-> Thr和Pro（-15）-> Leu仍然作为变性枯草杆菌蛋白酶BPN'的体外重折叠的伴侣蛋白起作用，与野生型前肽相比，效率分别为60%、80%和54%。对枯草杆菌蛋白酶BPN'的Ki值分别为1.6 × 10 ~（-9）M、1.2 × 10 ~（-9）M和2.1 × 10 ~（-9）M，几乎与野生型前肽显示的Ki值（1.4 × 10 ~（-9）M）相同。相比之下，Ile（-67）-->瓦尔和Gly（-44）--> Asp能够以仅18%和13%的效率重折叠变性枯草杆菌蛋白酶BPN'，并且具有分别为10和11 × 10（-9）M的Ki值。Ile（-48）--> Thr突变体前肽不能再折叠变性枯草杆菌蛋白酶BPN'，并给出比野生型前肽高100倍的Ki（118 × 10（-9）M）。从Leu（-19）延伸至Met（-78）的N59-mer前肽不能作为伴侣蛋白发挥功能。像野生型前肽，没有一个突变体前肽的二级结构，判断其圆二色谱。目前的结果表明，前肽作为伴侣蛋白的变性蛋白质的再折叠的能力是很好的相关性与其作为活性酶的竞争性抑制剂的能力。这支持了前肽的二级和三级结构在复性的前肽-枯草杆菌蛋白酶复合物和前肽与活性酶之间形成的抑制性复合物之间是相同的或高度同源的概念。
The amino-terminal propeptide, consisting of 77 amino acid residues, is known to be required as an in tramolecular 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.