Holt A, Alton G, Scaman C H, Loppnow G R, Szpacenko A, Svendsen I, Palcic M M
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
Biochemistry. 1998 Apr 7;37(14):4946-57. doi: 10.1021/bi972923l.
Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.
哺乳动物氨基脲敏感胺氧化酶（SSAO）已被归类为EC 1.4.3.6 [胺：氧氧化还原酶（脱氨基）（含铜）]。然而，醌辅因子的身份和铜的存在仍未得到证实，并且已证明无法将SSAO以足够的产量纯化至同质状态以进行辅因子鉴定。为了解决这个问题，我们从牛和猪的主动脉中部分纯化了SSAO酶，并通过氧化还原循环测定法确定酶制剂中不存在其他醌蛋白。然后用（对硝基苯基）肼（p-NPH）对酶进行衍生化，其与醌辅因子形成共价黄色复合物。衍生化的牛和猪酶的可见吸收光谱（在中性pH下分别为456和476 nm的最大波长，在2 M KOH中移至580和584 nm）与作为辅因子的（2,4,5-三羟基苯基）丙氨酸醌（TPQ）的存在一致。共振拉曼光谱与豌豆幼苗胺氧化酶（一种已知含TPQ的酶）的光谱基本相同。用链霉蛋白酶E对SSAO酶和猪肾二胺氧化酶进行广泛消化，得到具有二肽TPQ（p-NPH）-Asp特征的相同发色特性的物质。猪SSAO的热解消化产生了两个含辅因子的肽，其包含TPQ共有序列Asn-X-Asp-Tyr-Tyr，其中X是对应于TPQ的空白循环。全酶的N端测序揭示了细胞外II型糖蛋白典型的跨膜区域。这些结果证实了TPQ在哺乳动物膜结合SSAO外切酶中的存在。
