Structural and kinetic determinants of protease substrates

Nat Struct Mol Biol. 2009 Oct;16(10):1101-8. doi: 10.1038/nsmb.1668. Epub 2009 Sep 20.


Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in alpha-helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biochemistry / methods
  • Caspase 3 / metabolism
  • Catalysis
  • Escherichia coli / enzymology
  • Kinetics
  • Molecular Conformation
  • Mutation
  • Peptide Hydrolases / chemistry*
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proteomics / methods*
  • Signal Transduction
  • Substrate Specificity


  • Peptide Hydrolases
  • Caspase 3