studies of inhibition of cysteine endo- and exopeptidases by cystatins A and C
Pavlova, Alona
(2003).
Mechanism of action of mammalian cystatins.
Diss. (sammanfattning/summary)
Uppsala :
Sveriges lantbruksuniv.,
Acta Universitatis Agriculturae Sueciae. Veterinaria, 1401-6257
; 154
ISBN 91-576-6379-3
[Doctoral thesis]
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Abstract
Mammalian cystatins are tight-binding, reversible inhibitors of potentially destructive cysteine proteases, both endogenous and exogenous. The N-terminal region residues, Ile2 and especially Pro3, and the second binding loop residues, Leu73 and to some extent Pro74, of the family 1 cystatin, cystatin A, were shown to be essential for effective inhibition of papain and cathepsins L and B. Both regions were found to act in endopeptidase inhibition primarily by anchoring the enzymes in long-lived complexes with the inhibitor. However, in inhibition of the exopeptidase, cathepsin B, the N-terminal region also contributes to the high association rate. As previously shown for cystatin C, stopped-flow kinetics demonstrated a two-step mechanism of cystatin A binding to cathepsin B, with a conformational change in the second, rate-limiting step. In contrast, binding of cystatins A and C to a cathepsin B variant with an increased mobility of the occluding loop that blocks the active-site cleft occurred in an apparent one-step reaction. The second step of cathepsin B binding by cystatins was therefore concluded to involve displacement of the occluding loop. Cystatin C was shown to be a much better displacer of this loop than cystatin A. New effective and specific inhibitors of cysteine proteases were engineered by grafting individual binding regions of more potent cystatins into cystatin A. Introduction of motifs from the second binding loops of cystatins B or C into the corresponding loop of cystatin A increased the affinity for papain by ~10-fold. Moreover, replacement of the N-terminal region of cystatin A by that of cystatin C resulted in the affinity for cathepsin B being increased ~15-fold, to a level higher than that of any natural cystatin known. The latter chimeric inhibitor was as effective as cystatin C in displacing the occluding loop of cathepsin B, the rate of this step being increased by ~100-fold. The N-terminal region of a cystatin was thus concluded to promote occluding loop displacement by appropriately positioning the binding loops of the inhibitor entering the active-site cleft of cathepsin B.
Authors/Creators: | Pavlova, Alona | ||||
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Title: | Mechanism of action of mammalian cystatins | ||||
Subtitle: | studies of inhibition of cysteine endo- and exopeptidases by cystatins A and C | ||||
Year of publishing : | May 2003 | ||||
Number: | 154 | ||||
Number of Pages: | 64 | ||||
Papers/manuscripts: |
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Place of Publication: | Uppsala | ||||
ISBN for printed version: | 91-576-6379-3 | ||||
ISSN: | 1401-6257 | ||||
Language: | English | ||||
Publication Type: | Doctoral thesis | ||||
Full Text Status: | Public | ||||
Agris subject categories.: | X Agricola extesions > X30 Life sciences | ||||
Subjects: | Not in use, please see Agris categories | ||||
Agrovoc terms: | molecular biology, inhibition, enzyme inhibitors | ||||
Keywords: | cysteine protease, papain, cathepsin, inhibition, cystatin, affinity, binding kinetics, two-step reaction mechanism, occluding loop, binding loop | ||||
URN:NBN: | urn:nbn:se:slu:epsilon-57 | ||||
Permanent URL: | http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-57 | ||||
ID Code: | 300 | ||||
Department: | (VH) > Dept. of Molecular Biosciences (until 070101) | ||||
Deposited By: | Staff Epsilon | ||||
Deposited On: | 03 Jun 2003 00:00 | ||||
Metadata Last Modified: | 02 Dec 2014 10:03 |
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