The structure and function of ribonuclease T1. 8. Reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophan residue in ribonuclease T1.
The structure and function of ribonuclease T1. 8. Reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophan residue in ribonuclease T1.
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核糖核酸酶T1的结构和功能。
DOI：
10.1093/oxfordjournals.jbchem.a129279
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发表时间：
1970
影响因子：
2.7
通讯作者：
Kenji Takahashi
中科院分区：
文献类型：
--
作者：
Kenji Takahashi
1. 2-羟基-5-硝基苄基溴与核糖核酸酶Tj [EC 2. 7. 7. 26]为了获得关于59位上的单个色氨酸残基在该酶的功能中的作用的一些信息，已经对该酶进行了研究。在pH5.0或3.5和25 ℃下，在存在和不存在8 M尿素的情况下，用280- 1，000倍摩尔过量的试剂处理核糖核酸酶T（0.08 - 0.29%溶液）。2.在无尿素存在下，pH5.0处理30 min后，酶活力仍保持在90%以上，无明显反应发生。这表明酶中的色氨酸残基-59被埋在蛋白质分子的内部或与其他一些残基结合，因此变得不反应。3.当反应在pH5.0或3.5的8 U尿素存在下进行时，反应进行得非常迅速，并且对RNA和2 ′，3 ′-环鸟苷酸的酶活性同时丧失。这种失活几乎导致每分子2-羟基-5-硝基苄基中有一个残基掺入色氨酸残基的吲哚环中。在pH3.5条件下制备的最终产物几乎没有活性，但每分子含有近两个2-羟基-5-硝基苄基残基。4.失活蛋白的酸水解产物的氨基酸组成与天然蛋白的氨基酸组成没有区别。这表明反应特异性地与酶中的色氨酸残基发生。含有2-羟基-5-硝基苄基的Nagarse肽级分的分析结果支持这一结论。5.的反应性;通过用该试剂修饰色氨酸残基，在58位上邻近的谷氨酸残基的--羧基朝向碘乙酸的方向被显著地减少。6.这些证据表明，核糖核酸酶Ti中的色氨酸残基-59可能不直接参与催化过程，但很可能是构建酶活性中心结构的重要元素之一。
1. The reaction of 2-hydroxy-5-n itrobenzyl bromide with ribonuclease Tj [EC 2. 7. 7. 26] has been investigated in order to obtain some information on the role of the single tryptophan residue at position 59 in the function of this enzyme. Ribonuclease T, (0.08 to 0.29% solution) was treated with 280- to 1,000-fold molar excess of the reagent at pH5.0 or 3.5 and 25°C in the presence and absence of 8 M urea. 2. In the absence of urea, no significant reaction took place and the enzyme retained over 90% of the original activity after treatment at pH5.0 for 30min. This indicates that the tryptophan residue-59 in the enzyme is buried in the interior of the protein molecule or bound to some other residue or residues, thus becoming unreactive. 3. The reaction took place very rapidly when the reaction was carried out in the presence of 8u urea at pH5.0 or 3.5 and the enzymatic activity toward both RNA and 2', 3'-cyclic guanylic acid was lost in parallel. The inactivation almost paralleled the incorporation of one residue per molecule of 2-hydroxy-5-n itrobenzyl group into the indole ring of the tryptophan residue. The final product prepared at pH3.5 which wai almost devoid of activity, however, contained nearly two residues of 2-hydroxy-5nitrobenzyl group per molecule. 4. The amino acid composition of acid hydrolysates of the inactivated protein was indistinguishable from that of the native protein. This indicates that the reaction took place specifically with the tryptophan residue in the enzyme. The results of analysis of a Nagarse peptide fraction containing 2-hydroxy-5-nitrobenzyl group are in support for this conclusion. 5. The reactivity of the ;--carboxyl group of the adjacent glutamic acid residue at position 58 toward iodoacetate was markedly diminished by the modification of the tryptophan residue with the reagent. 6. These lines of evidence indicate that the tryptophan residue-59 in ribonuclease Ti is probably not directly involved in the catalytic process, but is very likely to be one of the important elements in building up the architecture of the active center of the enzyme.