Mechanism of Quinonoid Cofactor Formation in Copper Amine Oxidase and Catalytic Mechanism Involving Radical Intermediates

铜胺氧化酶催化各种生物胺的氧化脱胺反应，广泛存在于微生物、植物和动物中。除了铜以外，该酶还含有最近发现的一种喹酮辅因子--2，4，5-三羟基苯丙氨基苯醌(TPQ)，它与蛋白质共价结合。在本研究中，从一株科林型全球节杆菌中克隆了苯乙胺氧化酶和组胺氧化酶的编码基因，并进行了序列测定。利用在E.COIL细胞中过度生产的重组辅酶，已经证明了TPQ辅因子是由特定的酪氨酸残基通过蛋白质与二价铜离子结合而自我加工而产生的。用UV-Vis、CD、ESR和共振拉曼光谱详细研究了TPQ的形成过程，结果表明，与辅酶结合的铜离子首先被还原为亚铜离子，亚铜离子可能激活氧对前体酪氨酸残基进行氧化修饰，即半奎宁…在TPQ的形成过程中形成了一个以上的自由基中间体，并且TPQ的羰基C-2氧来源于溶剂。为了阐明保守序列Ans-Tyr-Asp/Glu-Tyr在TPQ生物发生中的作用(S)，通过定点突变，将重组组胺氧化酶第401-404位的相应残基分别替换为其他氨基酸。当ASN-401被改变为Asp或Gln时，依赖铜的自我加工形成TPQ的速度比野生型酶慢10^3~10^4倍。当用Phe取代Tyr-402时，没有形成TPQ，说明Tyr-402是TPQ的前体。相反，Asp-403可以被Glu取代而不改变TPQ的形成速度，而Asn则导致TPQ的生成显著减少。这些结果共同表明，有效地形成TPQ和铜胺氧化酶活性中心的催化活性需要非常严格的结构基序。测定了苯乙胺氧化酶的晶体结构，并对非活性脱氧核糖核酸和活性脱氧核糖核酸的晶体结构进行了精炼。两种形式之间的显著差异仅限于前体Tyr和TPQ的活性部位(位置和方向)以及与铜结合的His残基，表明在TPQ生物发生过程中蛋白质没有发生大的构象变化。较少

Copper amine oxidase catalyzes oxidative deamination of various biogenic amines, occurring widely in microorganisms, plants and animals. Besides copper, the enzyme contains a recently identified quinonoid cofactor, 2,4,5-trihydroxyphenylalanyl quinone (TPQ), covalently bound to the protein. In this study, the genes coding for phenylethylamine oxidase and histamine oxidase have been cloned from a Coryne-form bacterium Arthrobacter globiformis and sequenced. Using the recombinant apoenzyme overproduced in E.coil cells, the TPQ cofactor has been demonstrated to be produced from a specific tyrosyl residue by self-processing of the protein upon binding divalent copper ions. The process of TPQ formation has been investigated in detail by UV-VIS,CD,ESR,and resonance Raman spectroscopies to show that the cupric ion bound to the apoenzyme is first reduced to a cuprous ion, that the cuprous ion likely activates dioxygen for oxidative modification of the precursor tyrosyl residue, that a semiquin … More one radical intermediate is formed during the TPQ formation, and that the carbonyl C-2 oxygen of TPQ is derived from solvent. To elucidate the role(s) of the conserved sequence Ans-Tyr-Asp/Glu-Tyr in the biogenesis of TPQ,each of the corresponding residues at positions 401-404 in the recombinant histamine oxidase has been replaced with other amino acids by site-directed mutagenesis. When Asn-401 was changed to Asp or Gln, the rate of TPQ formation by copper-dependent self-processing was 10^3-to 10^4-fold slower than in the wild-type enzyme. When Tyr-402 was replaced by Phe, TPQ was not formed at all, showing that Tyr-402 is essential as the precursor to TPQ.In contrast, Asp-403 could be replaced by Glu without changes in the rate of TPQ formation, whereas its replacement by Asn led to a marked decrease. These results collectively indicate that a very rigorous structural motif is required for efficient formation of TPQ and for the catalytic activity in the active site of copper amine oxidases. The crystal structures phenylethylamine oxidase have been determined and refined for the inactive apo and active holo forms both at 2.2 resolution. Significant differences between the two forms are limited to the active site (positions and orientations of the precursor Tyr and TPQ and a His residue binding copper, suggesting that there occurrs no large conformational changes of the protein during the TPQ biogenesis. Less