Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction.
Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction.
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在碳二亚胺-胺反应中选择性修饰溶菌酶天冬氨酸-101残基的特定碳二亚胺结合机制。
DOI：
10.1093/oxfordjournals.jbchem.a135619
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发表时间：
1986
影响因子：
2.7
通讯作者：
T. Imoto
中科院分区：
文献类型：
--
作者：
R. Kuroki;H. Yamada;T. Imoto
在 pH 5.0 和室温下，使用乙醇胺作为亲核试剂，研究了在 1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺盐酸盐 (EDC) 催化下，用胺亲核试剂选择性修饰鸡蛋清溶菌酶中的 Asp-101 的机制。在N-乙酰基-D-氨基葡萄糖(NAG)及其寡聚物[(NAG)n、n=2和3]存在下，在约90%溶菌酶形成复合物的条件下，Asp-101修饰溶菌酶的形成明显减少，但程度不同，即(NAG)3最多，NAG最差。当使用活性位点裂缝中的Trp-62被氧化为oxindolealanine（Ox-62溶菌酶）的溶菌酶衍生物代替天然溶菌酶时，Asp-101修饰衍生物的形成减少到大约一半，这与（NAG）2存在时的减少类似。另一方面，在 0.5 M NaCl 存在下，Asp-101 修饰的溶菌酶的形成显着增强。从这些观察结果可以得出结论，EDC 与溶菌酶的活性位点裂缝结合，特异性激活 Asp-101。 EDC与溶菌酶活性位点的亲和力部分归因于EDC与溶菌酶B亚位点处的Trp-62残基的疏水相互作用。 EDC 是一种羧基活化剂，与大多数直接产生最终产品的活性定点试剂不同。因此，EDC 的活性位点定向性质非常有用，因为它可以根据需要在溶菌酶的特定羧基上选择性地引入各种胺。
A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.