Pfiffner E, Lerch K
Biochemistry. 1981 Oct 13;20(21):6029-35. doi: 10.1021/bi00524a017.
The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation.
通过染料敏化光氧化研究了组氨酸残基作为嗜热栖热放线菌酪氨酸酶双核活性位点铜潜在配体的作用。结果表明，在亚甲基蓝存在下光照，全酶的酶活性不受影响；然而，在相同条件下对脱辅基酶进行辐照会导致其被Cu2+重新激活的能力逐渐丧失。这种光失活与组氨酸含量的降低同时发生，而全酶中组氨酸残基的数量保持不变。对光氧化、重构的脱辅基酶进行铜含量测定表明，由于九个组氨酸残基中的三个发生了光敏氧化，每摩尔酶失去了一个铜原子的结合。通过比较光氧化的全酪氨酸酶和脱辅基酪氨酸酶中含组氨酸肽段的相对产量，确定了它们的序列位置。所获得的数据表明组氨酸残基188、193和289优先被修饰，并表明它们构成了两个活性位点铜原子之一的金属配体。发现用钴取代铜可使组氨酸残基完全免受染料敏化光氧化的修饰。
