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Doctoral thesis, 2001

Mechanism of interaction of the mammalian cysteine protease inhibitors, cystatin A and B, with target proteases

Pol, Ewa;

Abstract

Human cystatin A was shown to bind rapidly and strongly to papain and cathepsin L, with Åj of 0.2-20 pM and £ass of -3-5x10^ whereas the affinities for actinidin and cathepsins B, C and H were weaker (Kj 1-40 nM). The inhibition of cathepsin B was ~100-fold slower than that of papain, i. e. fcass was ~104 M’^-s'1. The binding to papain was consistent with a one-step binding mechanism. An N-terminally truncated cystatin A variant had an appreciably reduced affinity for papain, indicating the importance of this region for interaction with cysteine proteases.

Mutations in the second hairpin loop of cystatin B, Leu-73—>Gly and His-75—>Gly, decreased the affinity for papain and cathepsins L, H and B to an extent suggesting that this region contributes 20-30 % of the binding energy of cystatin B to target enzymes. A mutation in the C-terminal end of cystatin B, Tyr-97-»Ala, similarly indicated that this end contributes 6-12 % of the binding energy to papain and cathepsins L and H but is of limited importance for cathepsin B binding. The increased fcjiss for the binding of the mutants to proteases suggests that the two regions are important for complex stability.

Human and bovine wild-type cystatin B were shown to have indistinguishable inhibitory properties towards cysteine proteases, binding tightly to the endopeptidases, papain and cathepsin L, and more weakly to the exopeptidases, cathepsins H and B. Mutation of the single Cys residue, Cys-3, in cystatin B showed that this residue is involved in the binding of the inhibitor to target enzymes. Cys-3 is most important for cathepsin B binding and for the bovine inhibitor.

Sequential truncation of four residues from the N-terminal end of cystatin B resulted in progressively impaired affinities for papain and cathepsins L, H and B. The highest affinity loss was caused by removal of Cys-3, showing that this residue is most important for the binding. The decreased affinities ofthe truncated cystatin B mutants for papain and cathepsin H were due mainly to an increased Åtfiss» indicating that the N-terminal region keeps the inhibitor anchored to these enzymes in the complexes.

Keywords

cysteine protease; cysteine protease inhibitor; papain; cathepsin; cystatin; enzyme kinetics; inhibition; recombinant protein; stefin

Published in

Acta Universitatis Agriculturae Sueciae. Veterinaria

2001, number: 103
ISBN: 91-576-5927-3
Publisher: Swedish University of Agricultural Sciences

Authors' information

Pol, Ewa
Swedish University of Agricultural Sciences, Department of Veterinary Medical Chemistry

UKÄ Subject classification

Medical Bioscience

URI (permanent link to this page)

https://res.slu.se/id/publ/117486