Contributions of individual residues in the N-terminal region of cystatin B (stefin B) to inhibition of cysteine proteinases.
The importance of individual residues in the N-terminal region of cystatin B for proteinase inhibition was elucidated by measurements of the affinity and kinetics of binding of N-terminally truncated, recombinant variants of the bovine inhibitor to cysteine proteinases. Removal of Met-1 caused an 8- to 10-fold lower affinity for papain and cathepsin B, decreased the affinity also for cathepsin L but only minimally affected cathepsin H affinity. Additional truncation of Met-2 further weakened the binding to papain and cathepsin B by 40-70-fold, whereas the affinity for cathepsins L and H was essentially unaffected. Removal of Cys-3 had the most drastic effects on the interactions, resulting in a further affinity decrease of approximately 1500-fold for papain, approximately 700-fold for cathepsin L and approximately 15-fold for cathepsin H; the binding to cathepsin B could not be assessed. The binding kinetics could only be evaluated for papain and cathepsin H and showed that the reduced affinities for these enzymes were predominantly due to increased dissociation rate constants. These results demonstrate that the N-terminal region of cystatin B contributes appreciably to proteinase inhibition, in contrast to previous proposals. It is responsible for 12-40% of the total binding energy of the inhibitor to the proteinases investigated, being of least importance for cathepsin H binding. Cys-3 is the most important residue of the N-terminal region for inhibition of papain, cathepsin L and cathepsin H, the role of the other residues of this region varying with the target proteinase.
Pol
Ewa
E
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23, Uppsala, Sweden.
Björk
Ingemar
I
D000818:Animals; D002403:Cathepsins / Q000378:metabolism; D002417:Cattle; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010206:Papain / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D055313:Cystatin B; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D010206:Papain