Design of sensitive fluorogenic substrates for human cathepsin D

FEBS Letters

Majer, Pavel¹  Johnson, Donald G.²  Collins, Jack¹  Suvorov, Leonid I.¹  Gulnik, Sergei V.¹  Kane, Bradley P.²  Erickson, John W.¹ 

[1] Structural Biochemistry Program, SAIC Frederick, National Cancer Institute – Frederick Cancer Research and Development Center, Building 322, P.O. Box B, Frederick, MD 21702-1201, USA;AIDS Vaccine Program, SAIC Frederick, National Cancer Institute – Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA

关键词: Cathepsin D;    Fluorogenic substrate;    Structure-based modeling;    EDANS;    5-(2-aminoethyl) aminonaphthalene-1-sulfonic acid;    DABCYL;    4′-dimethylaminoazobenzene-4-carboxylic acid;    DIC;    diisopropyl carbodiimide;    DMF;    dimethylformamide;    DMSO;    dimethylsulfoxide;    Fmoc;    9-fluorenylmethoxycarbonyl;    HBTU;    2-(1H-benzotriazol-1-yl) 1;    1;    3;    3-tetramethyluronium hexafluorophosphate;    HOBt;    N-hydroxybenzotriazole;    NMP;    N-methyl-2-pyrrollidone;    PAL;    5-(4-aminomethyl-3;    5-dimethoxyphenoxy) pentanoic acid;    TFA;    trifluoroacetic acid;   

DOI  :  10.1016/S0014-5793(97)00886-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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/K _m 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.

【 授权许可】

Unknown