Cloning and characterization of a bifunctional leukotriene A4 hydrolase from Saccharomyces cerevisiae
Cloning and characterization of a bifunctional leukotriene A4 hydrolase from Saccharomyces cerevisiae
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DOI：
10.1074/jbc.274.49.34683
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发表时间：
1999-12-03
影响因子：
4.8
通讯作者：
Haeggström, JZ
中科院分区：
文献类型：
--
作者：
Kull, F;Ohlson, E;Haeggström, JZ
在哺乳动物中，白三烯A（4）水解酶是一种双功能锌金属酶，其催化白三烯A（4）水解为促炎性白三烯B-4，并且还具有乙酰氨基肽酶活性。我们已经从酿酒酵母中克隆、表达并鉴定了一种与人白三烯A（4）水解酶有42%相同的蛋白质。纯化的蛋白质是阴离子活化的亮氨酰氨肽酶，通过对硝基苯胺底物评估，并且不会将白三烯A（4）水解成可检测量的白三烯B-4。然而，S.酿酒酵母酶可以利用白三烯A作为底物来产生被鉴定为5S，6S-二羟基-7，9-反式-11，14-顺式-二十碳四烯酸的化合物。这两种催化活性均被3-（4-苄氧基苯基）-2-（R）-氨基-1-丙硫基乙胺（硫代胺）抑制，后者是人白三烯A（4）水解酶的竞争性抑制剂。此外，S.酿酒酵母酶被白三烯A刺激约10倍（4），动力学表明存在脂质结合位点。白三烯A（4）、白三烯B-4、花生四烯酸或磷脂酰胆碱的非酶水解产物无影响。此外，白三烯A（4）可取代抑制剂硫代胺并恢复最大氨肽酶活性，表明白三烯A（4）结合位点位于酶的活性中心。因此，S.酿酒酵母白三烯A（4）水解酶是一种双功能酶，似乎是哺乳动物白三烯A（4）水解酶的早期祖先。
In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B-4 and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B-4. However, the S. cerevisiae enzyme can utilize leukotriene A, as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B-4, arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A, binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.