Activation Mechanism of MelB Tyrosinase from Aspergillus oryzae by acidic treatment
Activation Mechanism of MelB Tyrosinase from Aspergillus oryzae by acidic treatment
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酸处理米曲霉MelB酪氨酸酶的激活机制
DOI：
10.1007/s00775-012-0945-5
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
S.
中科院分区：
文献类型：
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作者：
Fujieda;N.;Murata;M.;Yabuta;S.;Ikeda;T.;Shimokawa;C.;Nakamura;Y.;Hata;Y.;and Itoh;S.
重组酪氨酸酶的前体没有催化活性，但酸处理（pH3.5左右）激活它，诱导酪氨酸酶活性。圆二色谱，凝胶过滤分析，和比色测定表明，pH 3.5左右的酸处理诱导的C-末端结构域覆盖的酶活性位点的构象的破坏。酸处理引起的这些结构变化可以打开酶活性位点的入口，用于底物掺入。为了比较由酸处理的酪氨酸酶与胰蛋白酶处理的酪氨酸酶的羟基化的机制，通过使用Clark型氧电极监测O2-消耗速率来进行酚酶活性的详细稳态动力学分析。结果清楚地表明，活化的酪氨酸酶的酚酶活性（酚羟基化）涉及亲电芳族取代机制，如蘑菇酪氨酸酶的情况（Yamazaki和Itoh in J. Am. 125：13034-13035，2003）和用尿素活化的血蓝蛋白（Morioka等人，inJ. Chem. Am. 128：6788-6789，2006）。
The pro form of recombinant tyrosinase fromAspergillus oryzae(melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O2-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh inJ. Am. Chem. Soc.125:13034–13035, 2003) and activated hemocyanin with urea (Morioka et al. inJ. Am. Chem. Soc.128:6788–6789, 2006).