Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

J Biol Chem. 2006 May 5;281(18):12824-32. doi: 10.1074/jbc.M513331200. Epub 2006 Mar 6.


The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.

Publication types

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

MeSH terms

  • Animals
  • Binding, Competitive
  • Cathepsin K
  • Cathepsins / chemistry
  • Coumarins / chemistry
  • Cysteine / chemistry
  • Humans
  • Ketones / chemistry
  • Kinetics
  • Models, Chemical
  • Peptides / chemistry*
  • Phylogeny
  • Substrate Specificity


  • Coumarins
  • Ketones
  • Peptides
  • coumarin
  • Cathepsins
  • cathepsin S
  • CTSK protein, human
  • Cathepsin K
  • Cysteine