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Biochem. J. (1998) 335 (701–709) (Printed in Great Britain)

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Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases

Ingemar BJÖRK*¹, Kerstin NORDLING*, Elke RAUB-SEGALL*, Ulf HELLMAN† and Steven T. OLSON‡

*Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden, †Ludwig Institute for Cancer Research, Uppsala Biomedical Center, Box 595, SE-751 24 Uppsala, Sweden, and ‡Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, 801 S. Paulina Street, Chicago, IL 60612, U.S.A.

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.

¹ To whom correspondence should be addressed (e-mail Ingemar.Bjork@vmk.slu.se).

Received 21 May 1998/30 July 1998; accepted 1 September 1998

The Biochemical Society, London © 1998