Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes.
The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.
Björk
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I
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
Pol
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Raub-Segall
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Abrahamson
M
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Rowan
A D
AD
Mort
J S
JS
D000818:Animals; D002401:Cathepsin B / Q000378:metabolism; D002645:Chickens; D015891:Cystatins / Q000737:chemistry / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005361:Ficain / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010206:Papain / Q000378:metabolism; D011485:Protein Binding; D051381:Rats
D015891:Cystatins; C100158:cystatin, egg-white; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C018745:actinidain; D010206:Papain; D005361:Ficain