Galactose oxidase pro-sequence cleavage and cofactor assembly are self-processing reactions
Galactose oxidase pro-sequence cleavage and cofactor assembly are self-processing reactions
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DOI：
10.1021/ja993385y
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发表时间：
2000-02-09
影响因子：
15
通讯作者：
Dooley, DM
中科院分区：
文献类型：
--
作者：
Rogers, MS;Baron, AJ;Dooley, DM
半乳糖氧化酶是一个不断增长的蛋白质类的成员，具有新的后修饰的氧化还原活性氨基酸（见图1）。1这些修饰的不寻常性质激发了人们对产生这些辅因子的机制的兴趣。最近，胺氧化酶的2，4，5-三羟基苯丙氨酸醌（TPQ）辅因子的生物起源已被确定。2酪氨酸氧化为TPQ只需要铜离子和分子氧，不依赖于任何辅助蛋白。[3]半乳糖氧化酶的类似实验由于缺乏足够数量的纯前体（未加工的、无铜的）蛋白质而受到阻碍。在这里，我们报告的载脂蛋白，前酶形式的半乳糖氧化酶的分离，并证明，前序列的切割和组装的特征Tyr·-Cys辅因子是自我加工反应。图1说明了半乳糖氧化酶活性位点的关键特征。在氧化（活性）状态下，酪氨酸272通过硫醚键与半胱氨酸228后交联，被氧化成自由基。因此，[Cu（II）Tyr·-Cys]单元在各种醇氧化成相应的醛中充当双电子受体。翻译后交联被认为调节酪氨酰基自由基的反应性和氧化还原电位。5，6 C228可借助于硫原子的供电子性质而有助于自由基的稳定。5半乳糖氧化酶的氧化形式显示出一组特征性的电子跃迁（维达下文），这些电子跃迁也在乙二醛氧化酶（一种半乳糖氧化酶同系物）中观察到。7在铜限制条件下，构巢曲霉中镰刀菌蛋白的异源表达8导致多种蛋白形式的出现（图2）。图2a中SDS-PAGE条带的分子量估计为70.2、68.5和1065.5 kDa，通过蛋白质印迹法确定为半乳糖氧化酶。N-末端测序确定迁移最快的蛋白质（较低条带，约65.5 kDa）对应于成熟的野生型半乳糖氧化酶。成熟的半乳糖氧化酶在SDS-PAGE上以异常的分子量（65.5 kDa，与序列预测的68.5 kDa相比）迁移，这是由于硫醚键，其产生稳定的环，从而防止在用SDS处理时完全解折叠。8中间条带（图2a）的估计Mr与成熟半乳糖氧化酶氨基酸序列的质量相关，表明它是一种缺乏硫醚键的半乳糖氧化酶。这种行为被变体C228 G反映，其不能产生硫醚键。8最后，估计Mr为70.2 kDa的上部条带（图2a）对应于连接有前序列的前形式，这通过N-末端序列数据（表1）得到证实。通过在严格无金属的条件下进行生物体的生长和蛋白质纯化，首次实现了未加工半乳糖氧化酶的同质形式的纯化（图2b）。9添加Cu（II）和有氧孵育导致转化为成熟形式，如通过SDSPAGE监测的（图2b）。这表明前序列裂解和硫醚键形成都是铜介导的反应。前序列的切割不是由于外源性或内在蛋白酶活性，因为在与Cu（II）孵育期间存在蛋白酶抑制剂混合物（Sigma P-8215）时发生反应。重要的是，这款鸡尾酒...
Galactose oxidase is a member of a growing class of proteins with novel posttranslationally modified redox-active amino acids (see Figure 1). 1 The unusual nature of these modifications has stimulated interest in the mechanisms by which such cofactors are generated. Recently, the biogenesis of the 2, 4, 5-trihydroxyphenylalanine quinone (TPQ) cofactor of amine oxidase has been defined. 2 The oxidation of tyrosine to TPQ requires only copper ions and dioxygen, and is not dependent on any accessory proteins. 3 Analogous experiments with galactose oxidase have been hampered by the lack of sufficient quantities of pure precursor (unprocessed, copper-free) protein. Here we report the isolation of an apo, pro-enzyme form of galactose oxidase, and demonstrate that cleavage of the pro-sequence and assembly of the characteristic Tyr•-Cys cofactor are self-processing reactions. Figure 1 illustrates the critical features of the galactose oxidase active site. 4 In the oxidized (active) state tyrosine 272, which is posttranslationally cross-linked to cysteine 228 via a thioether bond, is oxidized to a radical. Thus the [Cu (II) Tyr•-Cys] unit acts as a two-electron acceptor in the oxidation of a wide variety of alcohols to the corresponding aldehydes. The posttranslational cross-link is believed to modulate the reactivity and redox potential of the tyrosyl radical. 5, 6 C228 may aid in stabilization of the radical by virtue of the electron-donating properties of the sulfur atom. 5 The oxidized form of galactose oxidase displays a characteristic set of electronic transitions (vida infra) that are also observed in glyoxal oxidase, a galactose oxidase homologue. 7 Heterologous expression8 of the Fusarium protein in Aspergillus nidulans under copper-limited conditions resulted in the appearance of multiple protein forms (Figure 2). The molecular weights of the SDS-PAGE bands in Figure 2a, established to be galactose oxidase by Western blotting, 8 were estimated as 70.2, 68.5, and∼ 65.5 kDa. N-terminal sequencing established that the fastest migrating protein (lower band,∼ 65.5 kDa) corresponds to mature, wild-type galactose oxidase. Mature galactose oxidase migrates on SDS-PAGE with an anomalous molecular weight (65.5 kDa as compared to 68.5 kDa predicted by the sequence), owing to the thioether bond, which produces a stable loop thus preventing full unfolding on treatment with SDS. 8 The middle band (Figure 2a) has an estimated Mr that correlates with the mass of the mature galactose oxidase amino acid sequence, suggesting that it is a form of galactose oxidase lacking the thioether bond. This behavior is mirrored by the variant C228G, which is unable to generate a thioether bond. 8 Finally, the upper band (Figure 2a), having an estimated Mr of 70.2 kDa, corresponds to the pro-form with the pro-sequence attached, which was confirmed by the N-terminal sequence data (Table 1). These data suggest that prosequence cleavage and thioether bond formation are separable reactions in vivo.Purification of a homogeneous form of unprocessed galactose oxidase was achieved for the first time by performing growth of the organism and protein purification under strictly metal-free conditions (Figure 2b). 9 Addition of Cu (II) and aerobic incubation result in conversion to the mature form as monitored by SDSPAGE (Figure 2b). This suggests that both pro-sequence cleavage and thioether bond formation are copper-mediated reactions. Cleavage of the pro-sequence is not due to extraneous or intrinsic protease activity because the reaction occurred when a proteaseinhibitor cocktail (Sigma P-8215) was present during the incubation with Cu (II). Importantly, this cocktail …