J. Biol. Chem., Vol. 262, Issue 16, 7552-7558, 06, 1987

Antithrombin conformation and the catalytic role of heparin. I. Does cleavage by thrombin induce structural changes in the heparin-binding region of antithrombin?

CB Peterson and MN Blackburn

Heparin has been shown to exhibit lower affinity for the antithrombin- thrombin complex than for antithrombin alone (Carlstrom, A.-S., Lieden, K., and Bjork, I. (1977) Thromb. Res. 11, 785-797), suggesting that structural alterations in antithrombin may accompany its reaction with thrombin. The hydroxy-nitrobenzyl (HNB) group attached to a unique tryptophan has been used in the present study as an extrinsic probe for localization of conformational changes to the heparin-binding region within antithrombin III using immunochemical and spectral techniques. Site-specific modification of tryptophan-49 in antithrombin with the hydroxynitrobenzyl reagent blocks heparin binding to the protein and provides a chemical label in the heparin-binding region of the protein (Blackburn, M. N., Smith, R. L., Carson, J., and Sibley, C. C. (1984) J. Biol. Chem. 259, 939-941). Antibodies specific for the hydroxynitrobenzyl hapten, which bind to HNB-tryptophan-49 in antithrombin, were used to detect a change in conformation in the region of tryptophan-49 which occurs upon thrombin binding to antithrombin. This thrombin-induced structural change was also apparent from spectral perturbations which were detected with the environmentally sensitive HNB moiety. Thus, the HNB group was used as an immunochemical probe as well as a spectral reporter group to provide insight into an allosteric mechanism of control in the catalytic role of heparin. The thrombin-promoted alteration of the structure in the heparin-binding region is presumably responsible for recycling of heparin, allowing it to catalyze further reactions between antithrombin and thrombin.
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