Pedersen J Z, el-Sherbini S, Finazzi-Agrò A, Rotilio G
Department of Biology, University of Rome Tor Vergata, Italy.
Biochemistry. 1992 Jan 14;31(1):8-12. doi: 10.1021/bi00116a002.
Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.
用底物还原铜胺氧化酶会导致产生一种自由基，该自由基在厌氧条件下可通过电子自旋共振（ESR）和光谱学检测到。通过对6-羟基多巴胺（最近鉴定出的辅因子6-羟基多巴的类似物）的半醌进行研究，考察了该自由基的来源。6-羟基多巴胺自由基的ESR谱太窄，无法解释酶自由基信号；然而，在与伯胺自发反应后，通过调制展宽获得的超精细分裂和光谱宽度与氧化酶自由基物种所观察到的非常相似。这种效应归因于氮原子直接与芳环结构的共价结合，这表明胺氧化酶自由基是氨基-6-羟基多巴半醌。使用腐胺、尸胺、对-[(二甲氨基)甲基]苄胺和乙二胺得到了相同的ESR谱；这些氧化酶底物也给出了相同的酶自由基谱。通过同位素标记技术明确证明了辅因子与底物之间的相互作用：添加[15N2]乙二胺而非正常的14N标记化合物改变了酶自由基及其6-羟基多巴胺对应物的ESR谱。光谱学测量结果证实了这些结果；6-羟基多巴胺和氧化的6-羟基多巴胺分别给出了与还原和氧化胺氧化酶相同的光谱。6-羟基多巴胺自由基在440 nm处有一个尖锐峰；加入胺后，最大值移至460 nm，这与酶的情况相同。有人提出，铜胺氧化酶是酪氨酸自由基酶家族中混合底物-辅因子自由基的第一个例子。
