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

Pol, Ewa (2001). Mechanism of interaction of the mammalian cysteine protease inhibitors, cystatin A and B, with target proteases. Diss. (sammanfattning/summary) Sveriges lantbruksuniv., Acta Universitatis Agriculturae Sueciae. Veterinaria, 1401-6257
ISBN 91-576-5927-3
[Doctoral thesis]

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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.

Authors/Creators:Pol, Ewa
Title:Mechanism of interaction of the mammalian cysteine protease inhibitors, cystatin A and B, with target proteases
Series Name/Journal:Acta Universitatis Agriculturae Sueciae. Veterinaria
Year of publishing :2001
Number of Pages:66
Publisher:Swedish University of Agricultural Sciences
ISBN for printed version:91-576-5927-3
Publication Type:Doctoral thesis
Article category:Other scientific
Version:Published version
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 4 Agricultural Sciences > 403 Veterinary Science > Medical Bioscience
Keywords:cysteine protease, cysteine protease inhibitor, papain, cathepsin, cystatin, enzyme kinetics, inhibition, recombinant protein, stefin
Permanent URL:
ID Code:28372
Faculty:VH - Faculty of Veterinary Medicine and Animal Science
Department:(VH) > Department of Veterinary Medical Chemistry
Deposited By: SLUpub Connector
Deposited On:17 Jun 2022 07:25
Metadata Last Modified:17 Jun 2022 07:40

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