Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.
Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.
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酶连接肽中的螺旋形成作为合成反应的驱动力：α-珠蛋白半合成反应的示例。
DOI：
10.1021/bi00147a007
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Acharya,AS
中科院分区：
文献类型：
--
作者：
Roy,RP;Khandke,KM;Manjula,BN;Acharya,AS
1992年2月24日收到的摘要：-珠蛋白半合成反应，即在30% 1-丙醇存在下，由V8蛋白酶催化的-珠蛋白的互补片段a 1-30和031-141的连接，与先前研究的片段互补系统的不连续位点的蛋白酶催化剪接相比是不同的[Sahni et al.（1989）Biochemistry 28，5456]。珠蛋白的互补片段不表现出它们之间的非共价相互作用，即使在1-丙醇，用于促进珠蛋白半合成反应的有机共溶剂的存在下。此外，片段n31_14i的显著部分不有助于蛋白酶催化的剪接反应。通过V8蛋白酶连接nl-30和nl-31-40以产生nl-30，其方式与nl-30分别与nl-141或nl-31 - 7剪接以产生n-珠蛋白或nl-31 - 7的方式大致相同。n 1-30和n 31-40的等摩尔混合物在30% 1-丙醇（用于合成反应的介质）存在下不显示任何“络合”。剪接点，即Glu 30-Arg 31肽键，位于亲本蛋白的B-螺旋（残基20-35）的中间。大部分来自蛋白A-螺旋的残余物也可以从片段a1 -30缺失而不影响V8蛋白酶催化的剪接反应。V8蛋白酶在30%正丙醇存在下催化α 30和α 31^ 0的剪接，总收率为40%，与半合成全长珠蛋白的收率相当。在不存在有机共溶剂的情况下，链段A表现出非常少的η-螺旋构象。另一方面，在1-丙醇的存在下，显著量的η-螺旋构象被诱导为η 17 -40。然而，即使在有机共溶剂的存在下，在η 17-40的Glu30_Arg31肽键处的不连续性也完全消除了肽的η-螺旋构象。因此，我们假设，在有机共溶剂的存在下，连续系统（与不连续系统相比）的螺旋构象增加作为半合成反应的“构象陷阱”。与这一假设相一致，V8蛋白酶催化的α-珠蛋白和α 1 -47的合成从各自的互补片段在其他螺旋诱导有机共溶剂如三氟乙醇和2-丙醇的存在下顺利进行。这种构象陷阱假说的一个推论是，只要所用酶的特异性要求得到满足，剪接反应就不依赖于所用的蛋白酶。胰蛋白酶确实在1-丙醇的存在下催化a17 - 31的Arg 31与a17 -40的Met 32的连接以产生连续区段a17 -40。结果表明，在互补片段的混合物中通过酶催化产生的共价邻接代表具有高“n-螺旋构象倾向”的新生片段的组装。的
Revised Manuscript Received February 24, 1992 abstract: The-globin semisynthetic reaction, namely, the ligation of the complementary fragments of-globin, a 1-30 and 031-141, in the presence of 30% 1-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al.(1989) Biochemistry 28, 5456]. The complementary fragments of-globin do not exhibit noncovalent interaction between them even in the presence of 1-propanol, the organic cosolvent used to facilitate the-globin semisynthetic reaction. Besides, a significant portion of the fragment «3i_i4i does not contribute to the protease-catalyzed splicing reaction.«i_3o and «31-40 are ligated by V8 protease toyield «1^ 0 in much the same way as the splicing of «1-30 with either «31-141 or «3^ 7 to yield «-globin or «1^ 7, respectively. An equimolar mixture of «1-30 and «31-40 does not show any ‘complexation’in the presence of 30% 1-propanol, the medium used for the synthetic reaction. The splicing junction, ie, Glu30-Arg31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of theresidues from the A-helix of the protein could also be deleted from segment a 1-30 without influencing the V8 protease-catalyzed splicing reaction. V8protease catalyzed the splicing of-30 and «31^ 0 in thepresence of 30% 1-propanol with an overall yield of 40% that compares well with the yield of semisynthesis of full length-globin. Segment^ exhibits very little «-helical conformation in the absence of the organic cosolvent. On the other hand, in the presence of 1-propanol, a significant amount of «-helical conformation is induced into-40· However, the discontinuity at the Glu30_Arg31 peptide bond of «17-40 completely abolished the «-helical conformation of the peptide even in the presence of the organic cosolvent. Therefore, we hypothesize that the increased helical conformation of the contiguous system (compared to the discontiguous system) in the presence of organic cosolvent operates as a ‘conformational trap’of the semisynthetic reaction. Consistent with this hypothesis, V8 protease-catalyzed synthesis of-globin and of ai-47 from therespective complementary fragments proceeds smoothlyin the presence of other helix-inducing organic cosolvents, like trifluoroethanol and 2-propanol. A corollary to this conformational trap hypothesis is that the splicing reaction will be independent of the protease used as far as the specificity requirements of theenzyme used are satisfied. Trypsin indeed catalyzed the ligation of Arg31 of ai7_3i with Met32 of «32-40 in the presence of 1-propanol to generate the contiguous segment «17-40. The results establish that the covalent contiguity generated by enzymic catalysis in a mixture of the complementary segments represents an assembly of a nascent segment with high ‘«-helical conformationalpropensity’. The