Temporary inhibition of papain by hairpin loop mutants of chicken cystatin Distorted binding of the loops results in cleavage of the Gly9‐Ala10 bond

FEBS Letters

Machleidt, Werner²  Assfalg-Machleidt, Irmgard²  Auerswald, Ennes A.³  Nägler, Dorit K.³  Stubbs, Milton T.¹  Fritz, Hans³ 

[1] Max-Planck-Institut für Biochemie, D-82152 Martinsried bei München, Germany;Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der LMU München, D-80336 München, Germany;Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innerstadt der LMU München, D-80336 München, Germany

关键词: Cysteine proteinase inhibitor;    Papain;    Cathepsin L;    Chicken cystatin variant;    Inhibition kinetics;    Temporary inhibition;    Boc-;    t-butyloxycarbonyl-;    Bz-;    benzoyl-;    ΔV55;    AEF-[S1M;    M29I;    ΔV55;    M89L] chicken cystatin;    ΔV55-S56;    AEF-[S1M;    M29I;    ΔV55;    ΔS56;    M89L] chicken cystatin;    ΔP103-L105;    AEF-[S1M;    M29I;    M89L;    ΔP103;    ΔW104;    ΔL105] chicken cystatin;    ΔI102-Q107;    AEF-[S1M;    M29I;    M89L;    ΔI102;    ΔP103;    ΔW104;    ΔL105;    ΔN106;    ΔQ107] chicken cystatin;    E-64;    l-3-carboxy-2;    3-trans-epoxy-propionyl-leucylamido-(4-guanidino)butane;    -NHMec;    7-(4-methyl)-coumarylamide;    -NHPhNO2;    p-nitroanilide;    PPIV;    papaya proteinase IV (glycyl endopeptidase;    EC 3 4 22 25);    rCC;    AEF-[S1M;    M29I;    M89L] chicken cystatin;    (recombinant chicken cystatin);    Suc-;    succinyl-;    Z-;    benzyloxycarbonyl-;   

DOI  :  10.1016/0014-5793(95)00174-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Temporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicken cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing revealed that reappearance of papain activity is due to selective cleavage of the Gly⁹-Ala¹⁰ bond in the N-terminal binding area of the chicken cystatin variants, resulting in truncated inhibitors of lower affinity. Cleavage of the same bond by contaminating papaya proteinase IV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme-inhibitor complex with first order rate constants k _temp of 2.3 × 10⁻³ up to 5 × 10⁻¹ s⁻¹. A similar ratio was found for all mutants (0.7 × 10⁶–2.1 × 10⁶ s⁻¹·M⁻¹); it is almost identical with the ratio of the peptide substrate Z-Phe-Arg-NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N-terminal contact area in a way that covalent interaction of the Gly⁹-Ala¹⁰ bond with the active-site Cys residue of papain can occur and the bond is cleaved in a substrate-like manner.

【 授权许可】

Unknown