DESIGN OF SENSITIVE FLUOROGENIC SUBSTRATES FOR HUMAN CATHEPSIN-D

Cathepsin D is a lysosomal aspartic proteinase that has been implicate d in several pathological processes such as breast cancer and Alzheime r's disease, We designed and synthesized a number of quenched fluoroge nic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK( DABCYL)E-NH2, where X=cysteine, methylcysteine, ethylcysteine, tert-bu tylcysteine, carboxymethylcysteine, methionine, valine or isoleucine, Most of the fluorogenic substrates exhibited greater k(cat)/K-m ratios than the best cathepsin D substrates described so far, Differences in kinetic constants, which were rationalized using structure-based mode ling, might make certain substrates useful for particular applications , such as active site titrations or initial velocity determination usi ng a fluorescent plate reader, (C) 1997 Federation of European Biochem ical Societies.