Gitlin G, Bayer E A, Wilchek M
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Biochem J. 1990 Jul 15;269(2):527-30. doi: 10.1042/bj2690527.
The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin.
通过使用酪氨酸特异性试剂对硝基苯磺酰氟（Nbs-F），研究了酪氨酸在蛋清糖蛋白抗生物素蛋白和细菌蛋白链霉抗生物素蛋白的生物素结合位点中的作用。平均每摩尔抗生物素蛋白亚基约0.5摩尔酪氨酸残基的修饰导致生物素结合完全丧失。这表明抗生物素蛋白亚基中的单个酪氨酸残基（Tyr-33）直接参与生物素结合位点，并且其被Nbs修饰也消除了相邻亚基的结合特性。这表明蛋清蛋白的酪氨酸残基也可能有助于天然蛋白质结构的稳定。然而，在链霉抗生物素蛋白中，平均每摩尔亚基3摩尔酪氨酸残基的修饰才能使蛋白质的生物素结合活性完全失活，但在生物素存在下，每摩尔亚基仅1摩尔（平均）酪氨酸残基受到保护。Nbs修饰的链霉抗生物素蛋白和Nbs修饰的链霉抗生物素蛋白-生物素复合物的酶消化产物的高效液相色谱洗脱图谱之间的差异显示，未受保护的蛋白质中有另外两个含有Nbs修饰酪氨酸残基的馏分。这些残基，Tyr-43（主要馏分）和Tyr-54（次要馏分），似乎对链霉抗生物素蛋白的生物素结合位点有贡献。
