title: Design of sensitive fluorogenic substrates for human cathepsin D.
abstract: 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.
authors.affiliation: Structural Biochemistry Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702-1201, USA. gulnik@ncifcrf.gov
authors.forename: S V
authors.initials: SV
authors.lastname: Gulnik
authors.forename: L I
authors.initials: LI
authors.lastname: Suvorov
authors.forename: P
authors.initials: P
authors.lastname: Majer
authors.forename: J
authors.initials: J
authors.lastname: Collins
authors.forename: B P
authors.initials: BP
authors.lastname: Kane
authors.forename: D G
authors.initials: DG
authors.lastname: Johnson
authors.forename: J W
authors.initials: JW
authors.lastname: Erickson
mesh_terms: D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D009282:Naphthalenesulfonates; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
chemical_list: D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009842:Oligopeptides; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D002402:Cathepsin D