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

Anal Biochem. 2016 Sep 1:508:38-45. doi: 10.1016/j.ab.2015.08.019. Epub 2015 Aug 24.

Abstract

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.

Keywords: 20S Eukaryotic proteasome; Fluorescence; Internally quenched peptides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Combinatorial Chemistry Techniques
  • Humans
  • 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

Substances

  • Peptides
  • Trypsin
  • Proteasome Endopeptidase Complex