Specificity-directed design of a FRET-quenched heptapeptide for assaying thermolysin-like proteases.
Dispase
Enzyme assay
M4 metallopeptidase
Metalloprotease
Quenched fluorescent peptide
Thermolysin
Journal
Analytical biochemistry
ISSN: 1096-0309
Titre abrégé: Anal Biochem
Pays: United States
ID NLM: 0370535
Informations de publication
Date de publication:
01 09 2020
01 09 2020
Historique:
received:
10
03
2020
accepted:
08
06
2020
pubmed:
6
7
2020
medline:
7
2
2021
entrez:
6
7
2020
Statut:
ppublish
Résumé
Thermolysin (TL) is an industrially important zinc endopeptidase, and the prototype of the M4 family of metallopeptidases. The catalytic function of TL and its relatives is typically assessed using chromogenic or more sensitive fluorescent peptides, with the latter substrates relying on Förster resonance energy transfer (FRET). Here, we demonstrate that a FRET-quenched heptapeptide designed on the basis of the enzyme's substrate specificity (Dabcyl-FKFLGKE-EDANS) is efficiently cleaved by TL and dispase (a TL-like protease) in between the Phe3 and Leu4 residues. The specificity constants (determined at pH 7.4 and 25 °C) for TL and dispase (3.6 × 10
Identifiants
pubmed: 32622975
pii: S0003-2697(20)30358-4
doi: 10.1016/j.ab.2020.113826
pii:
doi:
Substances chimiques
Bacterial Proteins
0
Thermolysin
EC 3.4.24.27
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
113826Informations de copyright
Copyright © 2020 Elsevier Inc. All rights reserved.