Design of sensitive fluorogenic substrates for human cathepsin D

FEBS Lett. 1997 Aug 18;413(2):379-84. doi: 10.1016/s0014-5793(97)00886-7.


Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E-NH2, where X=cysteine, methylcysteine, ethylcysteine, tert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k(cat)/Km ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure-based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.

MeSH terms

  • Cathepsin D / chemistry
  • Cathepsin D / metabolism*
  • Fluorescent Dyes / chemical synthesis*
  • Fluorescent Dyes / metabolism
  • Humans
  • Hydrogen Bonding
  • Liver / enzymology
  • Models, Molecular
  • Naphthalenesulfonates
  • Oligopeptides / chemical synthesis*
  • Oligopeptides / metabolism
  • Protein Conformation
  • Spectrometry, Fluorescence / methods
  • Substrate Specificity
  • p-Dimethylaminoazobenzene / analogs & derivatives


  • Fluorescent Dyes
  • Naphthalenesulfonates
  • Oligopeptides
  • 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid
  • 4-(4-dimethylaminophenylazo)benzoic acid
  • p-Dimethylaminoazobenzene
  • Cathepsin D