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

[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.

[学科分类] 生物化学/生物物理

[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