IDENTIFICATION OF TOPAQUINONE AND ITS CONSENSUS SEQUENCE IN COPPER AMINE OXIDASES
IDENTIFICATION OF TOPAQUINONE AND ITS CONSENSUS SEQUENCE IN COPPER AMINE OXIDASES
复制标题
DOI：
10.1021/bi00163a025
复制
发表时间：
1992-12-08
期刊：
影响因子：
2.9
通讯作者：
KLINMAN, JP
中科院分区：
文献类型：
--
作者：
JANES, SM;PALCIC, MM;KLINMAN, JP
在一系列铜胺氧化酶中，活性位点辅助因子的性质和该结构两侧的氨基酸序列已被确定。对于来自猪血浆、猪肾和豌豆幼苗的酶，对苯基腙或对硝基苯基腙衍生物进行蛋白水解消化。热溶素处理导致相对较小的活性位点肽，这些肽已被Edman降解和共振拉曼光谱表征。多肽的共振拉曼光谱显示出相同的峰位和强度，相对于托喹酮的对硝基苯腙衍生物，托喹酮在每个例子中都是辅助因子。Edman降解多肽提供了活性位点序列，与以前测定的牛血清和酵母胺氧化酶进行比较。现有数据建立了Asn、Topa、Asp/Glu的一致序列。胰蛋白酶导致肽明显变长，这表明牛和猪血浆蛋白之间的序列高度同源（89%），而猪血清和细胞内胺氧化酶之间的序列显著降低（56%）。在豌豆幼苗和哺乳动物酶之间观察到较低的同一性程度（45%）。作为一种替代分离活性位点肽来鉴定托喹酮的方法，研究了完整蛋白的可见光谱。结果表明，天然酶的对硝基苯基腙衍生物、活性位点衍生肽和托喹酮模型具有相同的吸收行为，在中性pH （pH 7.2）和碱溶液（1-2 M KOH）中，吸收波长分别为457 ~ 463 nm和575 ~ 587 nm。这些光谱性质似乎是托喹酮所特有的，为完整酶中这种辅因子的存在提供了一种快速而简单的测试方法。利用这种方法，已证实含有托喹酮的酶类已扩大到包括来自羊血浆和鹰嘴豆幼苗的铜胺氧化酶。
The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes. Using this approach, the class of enzymes demonstrated to contain topaquinone has been expanded to include copper amine oxidases from sheep plasma and chick pea seedling.