Cross-class inhibition of cysteine proteinases by serpins differs from serpin inhibition of serine proteinases primarily in that no stable serpin–cysteine proteinase complex can be demonstrated. This difference in reaction mechanism was elucidated by studies of the inactivation of the cysteine proteinases, papain and cathepsin L, by the serpin antithrombin. The two proteinases were inactivated with second-order rate constants of (1.6±0.1)×103 and (8.6±0.4)×102 M-1·s-1 respectively. An antithrombin to papain inactivation stoichiometry of ∼ 3 indicated extensive cleavage of the inhibitor concurrent with enzyme inactivation, a behaviour verified by SDS/PAGE. N-terminal sequence analyses showed cleavage predominantly at the P2–P1 bond, but also at the P2´–P3´ bond of antithrombin. The papain band in SDS/PAGE progressively disappeared on reaction of the enzyme with increasing amounts of antithrombin, but no band representing a stable antithrombin–papain complex appeared. SDS/PAGE with 125I-labelled papain showed that the disappearance of papain was caused by cleavage of the enzyme into small fragments. These results suggest a mechanism in which papain attacks a peptide bond in the reactive-bond loop of antithrombin adjacent to that involved in serine proteinase inhibition. The reaction proceeds, similarly to that between serpins and serine proteinases, to form an inactive acyl-intermediate complex, although with the substrate pathway dominating in the papain reaction. In this complex, papain is highly susceptible to proteolysis and is degraded by still active papain, which greatly decreases the lifetime of the complex and results in liberation of fragmented, inactive enzyme. This model may have relevance also for the inactivation of physiologically or pathologically important cysteine proteinases by serpins.
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November 1998
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Research Article|
November 01 1998
Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases
Ingemar BJÖRK;
Ingemar BJÖRK
1
*Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden
1To whom correspondence should be addressed (e-mail Ingemar.Bjork@vmk.slu.se).
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Kerstin NORDLING;
Kerstin NORDLING
*Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden
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Elke RAUB-SEGALL;
Elke RAUB-SEGALL
*Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden
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Ulf HELLMAN;
Ulf HELLMAN
†Ludwig Institute for Cancer Research, Uppsala Biomedical Center, Box 595, SE-751 24 Uppsala, Sweden
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Steven T. OLSON
Steven T. OLSON
‡Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, 801 S. Paulina Street, Chicago, IL 60612, U.S.A.
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Publisher: Portland Press Ltd
Received:
May 21 1998
Revision Received:
July 30 1998
Accepted:
September 01 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 335 (3): 701–709.
Article history
Received:
May 21 1998
Revision Received:
July 30 1998
Accepted:
September 01 1998
Citation
Ingemar BJÖRK, Kerstin NORDLING, Elke RAUB-SEGALL, Ulf HELLMAN, Steven T. OLSON; Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases. Biochem J 1 November 1998; 335 (3): 701–709. doi: https://doi.org/10.1042/bj3350701
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