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A Fluorogenic Method for Measuring Enteropeptidase Activity: Spectral Shift in the Emission of GD4K-conjugated 7-amino-4-methylcoumarin

A Fluorogenic Method for Measuring Enteropeptidase Activity: Spectral Shift in the Emission of GD4K-conjugated 7-amino-4-methylcoumarin

Mal-Gi Choi, Eungyeong Lee, Hye-Shin Chung, Sei-Heon Jang, ChangWoo Lee

BMB Rep. 2011 Jul;44(7):458-61.

PMID: 21777516

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP26093312-A	7-Amino-4-methylcoumarin		26093-31-2	Price

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