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氨，广泛存在于微生物、植物和动物中。除了铜，该酶还含有最近鉴定的醌辅因子，2，4，5-三羟基苯丙氨酰醌（TPQ），共价结合到蛋白质上。本研究从棒状杆菌球形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。使用在大肠杆菌细胞中过量产生的重组脱辅基酶，已经证明TPQ辅因子通过结合二价铜离子后蛋白质的自加工从特定的酪氨酰残基产生。通过UV-VIS、CD、ESR和共振拉曼光谱详细研究了TPQ的形成过程，表明与脱辅基酶结合的二价铜离子首先被还原为亚铜离子，亚铜离子可能活化分子氧，使前体酪氨酰残基氧化修饰，半喹啉 ...更多信息 在TPQ形成过程中形成一个自由基中间体，TPQ的羰基C2氧来自溶剂。为了阐明保守序列Ans-Tyr-Asp/Glu-Tyr在TPQ生物发生中的作用，重组组胺氧化酶中401-404位的每个相应残基已通过定点突变被其它氨基酸取代。当Asn-401变成Asp或Gln时，铜依赖性自我加工形成TPQ的速率比野生型酶慢10^3 ~ 10^4倍。当Tyr-402被Phe取代时，TPQ完全不形成，表明Tyr-402是TPQ的前体，而Asp-403被Glu取代时，TPQ的形成速率没有变化，但被Asn取代时，TPQ的形成速率明显降低。这些结果共同表明，一个非常严格的结构基序是需要有效地形成TPQ和铜胺氧化酶的活性位点的催化活性。苯乙胺氧化酶的晶体结构已被确定和完善的非活性载脂蛋白和活性全息形式都在2.2分辨率。两种形式之间的显著差异仅限于活性位点（前体Tyr和TPQ的位置和取向以及结合铜的His残基），这表明在TPQ生物发生期间蛋白质没有发生大的构象变化。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