Simultaneous fluorescent monitoring of proteasomal subunit catalysis

Aya Wakata, Hsien Ming Lee, Philipp Rommel, Alexei Toutchkine, Marion Schmidt, David S. Lawrence

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

The proteasome, a multicatalytic protease, displays distinct chymotrypsin-like, caspase-like, and trypsin-like activities at three different subunits of the multimeric complex. Fluorescent substrates for each of these active sites have been described. However, since the fluorescent properties of these substrates are very similar, it is not possible to simultaneously monitor catalysis of two or more activities. We have developed a long wavelength ( λex = 600 nm, λem = 700 nm) fluorescent substrate for the chymotrypsinlike active site via a combinatorial library strategy. This peptide-based substrate is a highly selective proteasomal chymotrypsin-like sensor, as assessed by a series of proteasomal active site mutants in yeast cell lysates. A corresponding caged analog of the sensor has been prepared, which is resistant to proteolysis until activated by 349 nm light. The latter affords the opportunity to assess proteasomal activity with a high degree of temporal control. The distinct photophysical properties of the sensor allow the chymotrypsin-like activity to be simultaneously monitored during caspase-like or trypsin-like catalysis. We have found that chymotrypsin-like activity is enhanced in the presence of the trypsin-like substrate but reduced in the presence of caspase-like substrate. Furthermore, the chymotrypsin-like sensor hinders the activity of both the caspase- and trypsin-like active sites. Coincident monitoring of two catalytic active sites furnishes twothirds coverage of total proteasomal activity, which should provide the means to address if and how the distinct active sites of the proteasome influence one another during catalysis.

Original languageEnglish (US)
Pages (from-to)1578-1582
Number of pages5
JournalJournal of the American Chemical Society
Volume132
Issue number5
DOIs
StatePublished - Feb 10 2010

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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    Wakata, A., Lee, H. M., Rommel, P., Toutchkine, A., Schmidt, M., & Lawrence, D. S. (2010). Simultaneous fluorescent monitoring of proteasomal subunit catalysis. Journal of the American Chemical Society, 132(5), 1578-1582. https://doi.org/10.1021/ja907226n