Use of an inhibitor to identify members of the hormone-sensitive lipase family
Use of an inhibitor to identify members of the hormone-sensitive lipase family
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DOI：
10.1021/bi0613978
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发表时间：
2006-11-28
期刊：
影响因子：
2.9
通讯作者：
Abousalham, Abdelkarim
中科院分区：
文献类型：
--
作者：
Ben Ali, Yassine;Chahinian, Henri;Abousalham, Abdelkarim
激素敏感性脂肪酶（HSL）对从脂肪细胞中储存的三酰甘油中动员脂肪酸有重要作用，脂肪细胞是哺乳动物的主要能量来源。基于氨基酸序列比对和三维结构，该酶先前被发现是用于定义丝氨酸羧酸酯水解酶家族的合适模板。在本研究中，HSL家族成员的特征在于它们被5-甲氧基-3-（4-苯氧基苯基）-3H-[1，3，4]恶二唑-2-酮（化合物7600）抑制。该化合物抑制哺乳动物HSL以及其他HSL家族成员，例如来自嗜热真细菌Alicyclobacillus acidocaldarius的EST 2和来自超嗜热古菌Archaeoglobus fulgidus的AFEST。发现在相同的实验条件下，化合物7600不抑制不是HSL家族成员的各种羧酸酯水解酶。这些酶包括非脂解水解酶，如加利福尼亚电鳐乙酰胆碱酯酶和猪肝酯酶，以及脂解水解酶，如人胰脂肪酶、狗胃脂肪酶、嗜热脂肪酶和枯草芽孢杆菌利帕。当乙烯基酯用作底物时，HSL、AFEST和EST 2的残留活性随着孵育混合物中化合物7600浓度的增加而降低。孵育5分钟后酶活性降低至50%时的抑制剂浓度分别为70、20和15 nM（HSL、AFEST和EST 2）。用抑制剂处理EST 2和AFEST导致分子量增加，如通过进行基质辅助激光解吸电离飞行时间质谱分析所建立的。这种分子量的增加（其大致对应于抑制剂的分子量）表明已经形成了共价酶-抑制剂复合物。用抑制剂处理或未处理的AFEST的胰蛋白酶消化物的表面增强激光解吸电离飞行时间质谱分析显示，含有“GESAGG”的肽的分子量增加，这与与抑制剂形成共价复合物相容。
Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)- 3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.