Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

FEBS Letters

Machleidt, Werner³  Assfalg-Machleidt, Irmgard³  Auerswald, Ennes A.²  Nägler, Dorit K.²  Stubbs, Milton T.¹  Fritz, Hans² 

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

关键词: Cysteine proteinase inhibitor;    Cysteine proteinase;    Chicken cystatin variant;    Protein engineering;    Inhibition kinetics;    E-64;    l-3-carboxy-2;    3-trans-epoxy-propionyl-leucylamido-(4-guanidino)butane;    NH-Mec;    7-(4-methyl) coumaryl-amide;    Z;    benzyloxycarbonyl;    rCC;    AEF-[SIM;    M29I;    M89L] chicken cystatin;    (recombinant chicken cystatin);    ΔV55;    AEF-[S1M;    M29I;    ΔV55;    M89L] chicken cystatin;    ΔV55-S56;    AEF-[S1M;    M29I;    ΔV55;    ΔS56;    M89L] chicken cystatin;    loop2-KD2;    AEF-[S1M;    M29I;    M89L;    S101I;    I102D;    P103I;    W104Q;    L105;    N106R;    Q107I;    I108A] 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;    N-del 2;    [ΔS1-P11;    V12A;    P13E;    V14F;    D15M;    M29I;    M89L] chicken cystatin;   

DOI  :  10.1016/0014-5793(95)00175-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Five recombinant hairpin loop variants of chicken cystatin (ΔV55, ΔV55-S56, ΔP103-L105, ΔI102-Q107, loop2-KD2) were constructed by cassette mutagenesis, expressed in E. coli, purified to homogeneity, characterized by protein-chemical means and by their inhibitory properties. The variant forms, modified in two of the three postulated cysteine proteinase binding regions, were inhibitorily active. However, the equilibrium dissociation constants of the complexes between papain as well as human cathepsin B or L and the cystatin variants show a weaker affinity for all three enzymes compared with recombinant chicken cystatin. These results prove the contribution of both hairpin loops to complex formation with the three enzymes. Furthermore, the kinetic constants indicate discrete differences in the molecular mechanism of interaction between chicken cystatin and papain, cathepsin B, and cathepsin L. Inhibition of cathepsin L was much less affected than inhibition of papain or cathepsin B by the modifications achieved in the five variants. Remarkably, at high enzyme concentration (above 0.5 nM) inhibition of papain by these variants was ‘temporary’, that means, active papain was released from the enzyme-inhibitor complex within minutes to hours (compare [1]).

【 授权许可】

Unknown