Rinaldi A C, Porcu C M, Oliva S, Curreli N, Rescigno A, Sollai F, Rinaldi A, Finazzi-Agró A, Sanjust E
Istituto di Chimica Biologica, Università di Cagliari, Italy.
Eur J Biochem. 1998 Jan 15;251(1-2):91-7. doi: 10.1046/j.1432-1327.1998.2510091.x.
Copper amine oxidases utilize 2,4,5-trihydroxyphenylalanine quinone (topaquinone) as a cofactor in enzymatic catalysis. This cofactor is formed from a tyrosine residue through a self-catalytic mechanism with the participation of the copper ion at the active site. Although pathways have been postulated for topaquinone biogenesis, portions of this scheme are still unclear. We utilized 4-tert-butyl-derived models for the putative intermediates of topaquinone generation and studied the effect of Cu(II) and Zn(II) ions on each autoxidative step from dopa- to topaquinone-like compounds at physiological pH (7.4). Several polyvinyl-alcohol-based soluble resins bearing mono- and di-hydroxyphenolic moieties were also prepared, and their tendency to give hydroxyquinonic structures when incubated at alkaline pH values was investigated. Our results confirm (although indirectly) the formation of dopa and dopaquinone during topaquinone biosynthesis. Moreover, we collected evidence that, following the formation of dopa, the role of the active-site copper ion in topaquinone biogenesis would be limited to the catalysis of the two subsequent quinonization steps (i.e. from dopa to dopaquinone and from topa to topaquinone), thus disfavoring the possibility of a direct intervention of the metal ion in the hydroxylation of dopaquinone. In particular, Cu(II) was shown to influence deeply the autoxidation of 1,2,5-trihydroxy-4-tert-butylbenzene, used as model of topa, both increasing the reaction rate and changing its mechanism. The mechanistic implications of these findings for the biogenesis of topaquinone and its analogs at the active site of various amine oxidases are discussed.
铜胺氧化酶在酶催化过程中利用2,4,5-三羟基苯丙氨酸醌（topaquinone，topa醌）作为辅因子。该辅因子由酪氨酸残基通过自催化机制形成，活性位点的铜离子参与其中。尽管已经提出了topa醌生物合成的途径，但该方案的部分内容仍不清楚。我们利用4-叔丁基衍生的模型来模拟topa醌生成的假定中间体，并研究了Cu(II)和Zn(II)离子对在生理pH值（7.4）下从多巴到topa醌样化合物的每个自氧化步骤的影响。还制备了几种带有单羟基和二羟基酚基部分的基于聚乙烯醇的可溶性树脂，并研究了它们在碱性pH值下孵育时生成羟基醌结构的倾向。我们的结果（尽管是间接的）证实了在topa醌生物合成过程中多巴和多巴醌的形成。此外，我们收集到的证据表明，在多巴形成后，活性位点铜离子在topa醌生物合成中的作用将仅限于催化随后的两个醌化步骤（即从多巴到多巴醌以及从topa到topa醌），因此不支持金属离子直接干预多巴醌羟基化的可能性。特别是，Cu(II)被证明对用作topa模型的1,2,5-三羟基-4-叔丁基苯的自氧化有深远影响，既提高了反应速率又改变了其机制。讨论了这些发现对各种胺氧化酶活性位点上topa醌及其类似物生物合成的机制意义。
