Department of Biomedical Science, Daegu University, Gyeongsan, Korea.
BMB Rep. 2011 Jul;44(7):458-61. doi: 10.5483/BMBRep.2011.44.7.458.
Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys (D(4)K). The assay for enteropeptidase has utilized GD(4)K-conjugated 2-naphthylamine (GD(4)K-NA) as a fluorogenic probe over the last 30 years. However, no other D(4)K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of GD(4)K-conjugated 7-amino-4-methylcoumarin (GD(4)K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a K(M) of 0.025 mM and a k(cat) of 65 sec(-1) for GD(4)K-AMC, whereas it has a K(M) of 0.5 to 0.6 mM and a k(cat) of 25 sec(-1) for GD(4)K-NA. The optimum pH of GD(4)K-AMC hydrolysis was pH 8.0. Our data indicate that GD(4)K-AMC is more suitable as a substrate for enteropeptidase than GD(4)K-NA.
肠肽酶是一种由胰腺分泌的丝氨酸蛋白酶，可将无活性的胰蛋白酶原转化为有活性的胰蛋白酶。肠肽酶切割底物识别序列 Asp-Asp-Asp-Asp-Lys（D(4)K）的 C 末端。在过去的 30 年中，肠肽酶的测定一直利用 GD(4)K-缀合的 2-萘基胺（GD(4)K-NA）作为荧光探针。然而，尚未报道其他 D(4)K 缀合的肠肽酶荧光底物。此外，萘被认为对人类具有致癌性。在这项研究中，我们使用 GD(4)K 缀合的 7-氨基-4-甲基香豆素（GD(4)K-AMC）的发射光谱位移作为荧光方法来测量肠肽酶活性。动力学分析表明，肠肽酶对 GD(4)K-AMC 的 K(M)为 0.025 mM，k(cat)为 65 sec(-1)，而对 GD(4)K-NA 的 K(M)为 0.5 至 0.6 mM，k(cat)为 25 sec(-1)。GD(4)K-AMC 水解的最适 pH 值为 pH 8.0。我们的数据表明，GD(4)K-AMC 比 GD(4)K-NA 更适合作为肠肽酶的底物。
