Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration by release of loop anchoring through mutation of His110

FEBS Letters

Krupa, Joanne C¹  Abrahamson, Magnus³  Björk, Ingemar²  Pavlova, Alla²  Mort, John S¹ 

[1] Joint Diseases Laboratory, Shriners Hospital for Children and the Department of Surgery, McGill University, Montreal, Que., Canada H3G 1A6;The Biomedical Center, Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, P.O. Box 575, SE-751 23 Uppsala, Sweden;Department of Clinical Chemistry, University of Lund, University Hospital, SE-221 85 Lund, Sweden

关键词: Cysteine proteinase;    Cysteine proteinase inhibitor;    Cathepsin;    Cystatin;    Stopped-flow kinetics;    C29A-cathepsin B;    cathepsin B variant in which Cys29 is replaced by Ala;    DTT;    dithiothreitol;    H110A/C29A-cathepsin B;    C29A-cathepsin B variant in which His110 is replaced by Ala;    K d;    overall dissociation equilibrium constant;    K i;    inhibition constant;    k obs;    observed pseudo-first-order rate constant;    k off;    overall dissociation rate constant;    k on;    overall association rate constant;    SDS–PAGE;    sodium dodecyl sulphate–polyacrylamide gel electrophoresis;   

DOI  :  10.1016/S0014-5793(00)02337-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Cystatins A and C were both shown to inhibit cathepsin B by a two-step mechanism, involving an initial weak interaction followed by a conformational change. Disruption of the major salt bridge anchoring the occluding loop of cathepsin B to the main body of the enzyme by mutation of His110 to Ala converted the binding to an apparent one-step reaction. The second step of cystatin binding to cathepsin B must therefore be due to the inhibitor having to alter the conformation of the enzyme by displacing the occluding loop to allow a tight complex to be formed. Cystatin A was appreciably less effective in displacing the loop than cystatin C, resulting in a considerably lower overall inhibition rate constant.

【 授权许可】

Unknown