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Interaction of cystatins with cysteine proteinases

Nycander, Maria (1998). Interaction of cystatins with cysteine proteinases. Diss. (sammanfattning/summary) Sveriges lantbruksuniv., Acta Universitatis Agriculturae Sueciae. Veterinaria, 1401-6257
ISBN 91-576-5428-X
[Doctoral thesis]

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The single tryptophan residue, Trpl04, of the cysteine proteinase inhibitor, chicken cystatin, was modified with a 2-hydroxy-5-nitrobenzyl group. The change of the absorbtion spectrum on binding ofthe modified cystatin to papain indicated a decreased enviromental polarity of the probe. The modified inhibitor had a greatly reduced affinity for papain. These results show that Trpl04 of cystatin is located in or near the proteinasebinding site.Characterization ofN-terminally truncated forms of chicken cystatin indicated that the Nterminal region contributes approximately 40% of the total free energy change for the binding of the inhibitor to both papain and actinidin. Leu7 and Leu8 account for about two-thirds ofthis binding energy. Also the highly conserved Gly9 residue and residues Nterminal to Leu7 contribute binding energy, but to a much smaller extent.Cruzipain, the major cysteine proteinase from the parasite, Trypanosoma cruzi, was tightly and rapidly inhibited by cystatin A, B, C, chicken cystatin and kininogen. This shows that cruzipain can be effectively inhibited by host cystatins, and that cystatins may serve as starting points for the design ofinhibitors as antiparasite drugs.The affinity of Gly4 mutants of cystatin A for papain, cathepsin L and cathepsin B decreased with the size of the substituent. Even the smallest substitution, to Ala, reduced the affinity >1000-fold. For papain and cathepsin L the effect was entirely due to increased dissociation rate constants. In contrast, for cathepsin B the mutations affected both the association and dissociation rate constants, consistent with the N-terminal region of cystatin A serving as a guide in binding ofthe remainder ofthe inhibitor to cathepsin B.​​​​​​​Stopped-flow kinetics showed a hyperbolic concentration dependence of the observed pseudo-first-order rate constant for the binding of cystatin C to cathepsin B, indicating a two-step binding mechanism. The first step most probably involves an initial weak binding of the N-terminal region of cystatin C to cathepsin B, whereas the second step involves a conformational change due to the displacement of the occluding loop of the enzyme.

Authors/Creators:Nycander, Maria
Title:Interaction of cystatins with cysteine proteinases
Series Name/Journal:Acta Universitatis Agriculturae Sueciae. Veterinaria
Year of publishing :1998
Number of Pages:46
Publisher:Swedish University of Agricultural Sciences
ISBN for printed version:91-576-5428-X
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:proteinase, protease, peptidase, proteinase inhibitor, cysteine proteinase, cysteine proteinase inhibitor, papain, cathepsins, cruzipain, cystatins, stefins, inhibition, enzyme kinetics, two-step reaction mechanism
Permanent URL:
ID Code:28314
Faculty:VH - Faculty of Veterinary Medicine and Animal Science
Department:(VH) > Department of Veterinary Medical Chemistry
Deposited By: SLUpub Connector
Deposited On:13 Jun 2022 08:43
Metadata Last Modified:13 Jun 2022 08:57

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