ENGINEERING SUBTILISIN AND ITS SUBSTRATES FOR EFFICIENT LIGATION OF PEPTIDE-BONDS IN AQUEOUS-SOLUTION
ENGINEERING SUBTILISIN AND ITS SUBSTRATES FOR EFFICIENT LIGATION OF PEPTIDE-BONDS IN AQUEOUS-SOLUTION
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DOI：
10.1021/bi00231a007
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发表时间：
1991-04-30
期刊：
影响因子：
2.9
通讯作者：
WELLS, JA
中科院分区：
文献类型：
--
作者：
ABRAHMSEN, L;TOM, J;WELLS, JA
利用蛋白质工程技术构建了丝氨酸蛋白酶枯草菌素的衍生物，该衍生物能在水中有效地连接肽。催化Ser221转化为Cys (S221C)， Pro225转化为Ala （P225A）的枯草杆菌素双突变体比单突变的硫基枯草杆菌素（S221C）具有高10倍的肽连接酶活性，至少低100倍的酰胺酶活性，而单突变的硫基枯草杆菌素（S221C）先前被证明具有一些肽连接酶活性[Nakatsuka， T., Sasaki， T., & Kaiser， E. T.(1987)]。化学。[j].中国生物医学工程学报，2016,33(2):481 - 481。双突变体（S221C/P225A）的氧化衍生物的1.5埃x射线晶体结构支持蛋白质设计策略，表明P225A突变部分缓解了S221C取代所期望的空间拥挤，从而解释了其提高的催化效率。制备了稳定且合成合理的烷基酯肽底物，可快速使S221C/P225A酶酰化，并且各种肽对硫酰基酶中间体的氨基水解比水解更有优势。氨解的效率相对不敏感的前两个残基在酰基受体肽，其α -氨基攻击硫酰基酶的序列。为了在偶联位点的选择上获得更大的灵活性，通过在亲本连接酶（S221C/P225A）中引入突变，设计了一组额外的三个肽连接酶，这些突变先前被证明可以改变枯草杆菌素对最靠近酰基键的残基（P1残基）的特异性。亲本连接酶及其衍生物的特异性与野生型和相应的特异性变异相似。这组特殊的肽连接酶可用于水溶液中蛋白质的块合成或半合成。
Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E. T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-angstrom X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants. The set of specific peptide ligases should be useful for blockwise synthesis or semisynthesis of proteins in aqueous solution.