Specificity of α2-Macroglobulin Covalent Cross-Linking for the Active Domain of Proteinases

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Abstract
The reaction of .alpha.2-macroglobin (.alpha.2M) with the two-chain enzyme plasma kallikrein results in covalent bond formation between the catalytic subunit and the inhibitor. We have recently published a model of .alpha.2M which suggests that this phenomenon may be a general mechanism when multisubunit proteinases are inactivated by .alpha.2M. In order to test this hypothesis, we studied the reactions of factor Xa, plasmin, Streptokinase-plasmin and .alpha.-thrombin with .alpha.2M. In the case of factor Xa the catalytic heavy chain demonstrated greater than 99% covalent incorporation while over 97% of the light chain failed to crosslink to the inhibitor. Preferential binding of the catalytic light chains of plasmin (70% covalent incorporation) and plasmin in complex with streptokinase (79% covalent incorporation) was also observed. Finally, 82% covalent incorporation of the catalytic heavy chain of .alpha.-thrombin was found. These studies demonstrate that in the case of multisubunit proteinases, the chain containing the active site demonstrates preferential binding as predicted by the model supporting placement of the site of covalent binding close to the "bait region" of .alpha.2M.