Novel internally quenched substrate of the trypsin-like subunit of 20S eukaryotic proteasome

• Published in: Analytical biochemistry Vol. 508; pp. 38 - 45
• Main Authors: Gruba, Natalia, Wysocka, Magdalena, Brzezińska, Magdalena, Debowski, Dawid, Rolka, Krzysztof, Martin, Nathaniel I., Lesner, Adam
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2016
• Subjects: 20S Eukaryotic proteasome; Combinatorial Chemistry Techniques; Fluorescence; Humans; Internally quenched peptides; Limit of Detection; Peptides - antagonists & inhibitors; Peptides - chemical synthesis; Peptides - chemistry; Peptides - metabolism; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - metabolism; Proteolysis; Trypsin - chemistry; Trypsin - metabolism; ACC; SDS; DIPEA; Fluorescence; TBTU; 20S Eukaryotic proteasome; Internally quenched peptides; TFA; RP-HPLC; PR3; DIPCI; ANB; AMC; HOBt; DMAP; UPS; ABZ; FRET; Tyr(3-NO2); DMF
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2015.08.019

This article describes the synthesis, using combinatorial chemistry, of internally quenched substrates of the trypsin-like subunit of human 20S proteasome. Such substrates were optimized in both the nonprime and prime regions of the peptide chain. Two were selected as the most susceptible for proteasomal proteolysis with excellent kinetic parameters: (i) ABZ-Val-Val-Ser-Arg-Ser-Leu-Gly-Tyr(3-NO2)-NH2 (kcat/KM = 934,000 M−1 s−1) and (ii) ABZ-Val-Val-Ser-GNF-Ala-Met-Gly-Tyr(3-NO2)-NH2 (kcat/KM = 1,980,000 M−1 s−1). Both compounds were efficiently hydrolyzed by the 20S proteasome at picomolar concentrations, demonstrating significant selectivity over other proteasome entities.