AN INVESTIGATION OF BOVINE SERUM AMINE OXIDASE ACTIVE-SITE STOICHIOMETRY - EVIDENCE FOR AN AMINOTRANSFERASE MECHANISM INVOLVING 2 CARBONYL COFACTORS PER ENZYME DIMER
AN INVESTIGATION OF BOVINE SERUM AMINE OXIDASE ACTIVE-SITE STOICHIOMETRY - EVIDENCE FOR AN AMINOTRANSFERASE MECHANISM INVOLVING 2 CARBONYL COFACTORS PER ENZYME DIMER
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DOI：
10.1021/bi00232a034
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发表时间：
1991-05-07
期刊：
影响因子：
2.9
通讯作者：
KLINMAN, JP
中科院分区：
文献类型：
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作者：
JANES, SM;KLINMAN, JP
最近的证据表明，牛血清胺氧化酶（BSAO）的活性位点辅助因子是2,4,5-三羟基苯丙氨酸或6-羟多巴[Janes et al. (1990) Science 248, 981]。然而，关于酶促反应的机制仍有许多不明确之处。在二聚体酶的功能活性位点的数量和产物释放的氧依赖性方面存在矛盾的数据。为了解决这些问题，我们开发了一种新的纯化BSAO的方法，可以在2-3周内分离出比活性大于或等于0.4单位/mg的酶。这种高活性酶被用于定量还原半反应中醛和氨的释放。酶和底物厌氧孵育产生2 mol醛/mol酶，表明在每个酶亚基上存在一个辅因子。正如对转氨酶反应的预期，在可比条件下没有检测到氨释放。用苯肼对不同比活性的酶样品进行活性位点滴定，得到比活性为0.48单位/mg的BSAO的1 mol抑制剂/mol酶亚基。这些发现与先前报道的铜胺氧化酶中每个酶二聚体只有一个功能辅助因子形成对比。
Recent evidence has shown that the active site cofactor in bovine serum amine oxidase (BSAO) is 2,4,5-trihydroxyphenylalanine or 6-hydroxydopa [Janes et al. (1990) Science 248, 981]. However, much ambiguity remains regarding the mechanism of the enzymatic reaction. Conflicting data exist for both the number of functional active sites in the dimeric enzyme and for the oxygen dependence of product release. To resolve these questions, a new method has been developed for the purification of BSAO which leads to the isolation of specific activity greater-than-or-equal-to 0.4 unit/mg of enzyme in 2-3 weeks. This highly active enzyme has been used to quantitate both aldehyde and ammonia release in the reductive half-reaction. Anaerobic incubation of enzyme and substrate resulted in the production of 2 mol of aldehyde/mol of enzyme, indicating the presence of a cofactor at each enzyme subunit. As anticipated for an aminotransferase reaction, no ammonia release was detected under comparable conditions. Active site titration of enzyme samples of varying specific activity with phenylhydrazine extrapolates to 1 mol of inhibitor/mol of enzyme subunit for BSAO of specific activity = 0.48 unit/mg. These findings contrast with numerous, previous reports of only one functional cofactor per enzyme dimer in copper amine oxidases.