Wollmer A, Strassburger W, Glatter U, Dodson G G, McCall M, Gattner H G, Danho W, Brandenburg D, Rittel W
Hoppe Seylers Z Physiol Chem. 1981 Jun;362(6):581-91. doi: 10.1515/bchm2.1981.362.1.581.
In 1979 the first abnormal human insulin was discovered. With the minute samples from the patient a Phe leads to Leu replacement could be established in either position B24 or B25. For the unequivocal localization of the substitution both the Leu analogues had to be prepared by semisynthesis. While another laboratory did this with the sequence of porcine insulin, here we are dealing with the true analogues of human insulin. In the present paper the structural consequences of the substitutions are investigated. Human insulin obtained by total synthesis served as a reference. Its CD spectral properties are herewith documented. According to the substantial deviations of the CD spectrum of [Leu B24]insulin, the introduction of the new side-chain forces conformational changes to occur not only in its immediate surrounding but also in the peptide chain. The failure to give the typical CD spectral response to variations of protein and zinc concentration indicates that the ability to form quaternary structure is impaired. Though dimerization was confirmed by gel chromatography to be largely reduced, it is concluded that, in addition, interactions normally responsible for the increase in tyrosine-CD with association are weakened. [Leu B25]insulin, on the other hand, does exhibit all CD spectral effects characteristic of the native hormone, though quantitatively somewhat reduced. The CD spectroscopic results are in full agreement with the computergraphic analysis of the sterical consequences of the substitutions. For B24-leucine an acceptable packing without movements of the mainchain and/or B15-leucine and without affecting dimerization is impossible, whereas B25-leucine can be accommodated without causing bad contacts either in the monomer or in the dimer. The structural results do not explain why [Leu B25]-insulin should have a lower biological activity than the B24 analogue, 2.1 +/- 0.3% versus 20.9 +/- 2.8%, in the fat cell test. They suggest, however, an important but not critical stereospecific role for the B25-phenylalanine in binding.
1979年，第一种异常人胰岛素被发现。利用患者的微量样本，确定了在B24或B25位存在苯丙氨酸被亮氨酸取代的情况。为了明确取代位置，两种亮氨酸类似物都必须通过半合成制备。当另一个实验室用猪胰岛素序列进行此项工作时，我们这里处理的是真正的人胰岛素类似物。在本文中，研究了取代的结构后果。通过全合成获得的人胰岛素用作参考。其圆二色光谱特性在此记录。根据[亮氨酸B24]胰岛素圆二色光谱的显著偏差，新侧链的引入不仅使其紧邻区域发生构象变化，而且使肽链也发生构象变化。对蛋白质和锌浓度变化未能给出典型的圆二色光谱响应，表明形成四级结构的能力受损。尽管凝胶色谱法证实二聚化程度大幅降低，但可以得出结论，此外，通常与缔合相关的酪氨酸圆二色性增加所涉及的相互作用也减弱了。另一方面，[亮氨酸B25]胰岛素确实表现出天然激素所有的圆二色光谱效应，尽管在数量上有所降低。圆二色光谱结果与取代的空间后果的计算机图形分析完全一致。对于B24位亮氨酸，不可能在不移动主链和/或B15位亮氨酸且不影响二聚化的情况下实现可接受的堆积，而B25位亮氨酸在单体或二聚体中都可以容纳而不产生不良接触。结构结果无法解释为什么在脂肪细胞试验中，[亮氨酸B25]胰岛素的生物活性比B24类似物低，分别为2.1±0.3%和20.9±2.8%。然而，它们表明B25位苯丙氨酸在结合中起重要但非关键的立体特异性作用。
