Schmidinger Hannes, Birner-Gruenberger Ruth, Riesenhuber Gernot, Saf Robert, Susani-Etzerodt Heidrun, Hermetter Albin
Institut für Biochemie, Technische Universität Graz, Petersgasse 12/II, 8010 Graz, Austria.
Chembiochem. 2005 Oct;6(10):1776-81. doi: 10.1002/cbic.200500013.
Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)selective modification of synthetic substrates. Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations. The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis. The selectivities of the inhibitors were determined by using different (phospho)lipase, esterase and cholesterol esterase preparations. The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor. We identified inhibitors with very low specificity, and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases. A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.
脂肪酶和酯酶负责细胞内外的羧酸酯水解，是用于合成底物（立体）选择性修饰的有用生物催化剂。在此，我们描述了结构和极性不同的新型荧光自杀抑制剂，用于筛选和区分酶制剂中的脂解酶。这些抑制剂与酶发生共价反应，形成可通过凝胶电泳分离的荧光脂质 - 蛋白质复合物。通过使用不同的（磷酸）脂肪酶、酯酶和胆固醇酯酶制剂来确定抑制剂的选择性。结果表明，抑制剂 - 酶复合物的形成高度依赖于抑制剂的化学结构。我们鉴定出特异性非常低的抑制剂，以及对某些脂解酶亚组（如脂肪酶和胆固醇酯酶）具有高度特异性的其他衍生物。这些底物类似活性探针的组合是用于快速鉴定和分类丝氨酸水解酶的有用工具箱。
