Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen
Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen
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DOI：
10.1021/bi8010835
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发表时间：
2008-09-30
期刊：
影响因子：
2.9
通讯作者：
McPherson, Michael J.
中科院分区：
文献类型：
--
作者：
Rogers, Melanie S.;Hurtado-Guerrero, Ramon;McPherson, Michael J.
半乳糖氧化酶（GO）属于一类自催化活性位点氨基酸组装其氧化还原活性辅助因子的蛋白质。产生具有酶活性的氧化石墨烯似乎需要至少四个顺序的翻译后修饰：分泌信号序列的切割，n端前序列的铜依赖切割，C228-Y272硫醚键的铜依赖形成，以及Y272自由基的生成。最后两个过程使用缺乏前序列的截断蛋白（称为premat-GO）进行研究，并在无铜条件下纯化。利用光学、电子磁共振和共振拉曼光谱、SDS-PAGE和x射线晶体学研究了预氧化石墨烯与Cu（II）的反应。pre - go与过量的Cu（II）发生厌氧反应，有效地形成硫醚键，但不能形成Y272自由基。从吸收光谱中发现了一个尚未形成C228-Y272交联的潜在c228 -铜配位中间体（λ (max) = 406 nm）。在厌氧浸泡3分钟后，通过x射线晶体学观察到铜与C228、H496和H581配位的铜-硫酸盐蛋白复合物，而浸泡24小时后发现C228- y272硫醚键。在溶液中，将含氧缓冲液添加到与过量Cu（II）预孵育的氧化石墨烯中，生成Y272自由基态。在此基础上，提出了C228-Y272键形成和酪基自由基生成的机理。在厌氧条件下，406 nm的配合物被证明是一个具有催化能力的加工中间体。我们提出了一种与Cu（II）有氧处理相同的潜在机制，直到需要第二个电子受体的步骤。
Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redox-active cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (lambda(max) = 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required.