Oxidative dissociation of human α₂-macroglobulin tetramers into dysfunctional dimers

Human α₂-macroglobulin is a broad-spectrum, homotetrameric antiproteinase that can maximally bind up to two proteinase molecules in a ternary complex. Proteinases cleave the inhibitor within a peptide stretch termed the bait region and induce the emergence of internal thiol esters whose nucleophilic scission precede a major conformational change which entraps enzymes within molecular cages. In a previous study, leukocyte- generated hypohalous acids and N-haloamines were identified as the first examples of physiologically relevant inactivators of the antiproteolytic activity of α₂-macroglobulin (Reddy, V. Y., Pizzo, S. V., and Weiss, S. J. (1989) J. Biol. Chem. 264, 13801-13809), but the mechanisms whereby the oxidants damaged the inhibitor remained undefined. We now demonstrate that N- chloramines (RNCl) destroy the antiproteolytic activity of α₂-macroglobulin in an unusual biphasic process that results in the formation of inactive α₂-macroglobulin half-molecules. In the first phase, 8 eq of RNCl reacted with each α₂-macroglobulin subunit to generate a partially oxidized antiproteinase containing 8 methionyl sulfoxide residues/monomer. Structure- function analyses demonstrated that the oxidized inhibitor retained its homotetrameric structure as well as its ability to entrap proteinases. In marked contrast, the oxidation of an additional 6 methionyl residues and a single tryptophanyl residue fractured the α₂M homotetramer across its non- covalent axis into two pairs of disulfide-linked dimers. Despite the fact that the oxidized dimers displayed normal bait regions whose cleavage by proteinases initiated thiol ester scission, all antiproteolytic activity was lost. Furthermore, the oxidized dimers were unable to undergo the critical conformational changes normally associated with bait region cleavage or thiol ester scission. Together, these results demonstrate that chlorinated oxidants destroy the antiproteolytic activity of α₂-macroglobulin by attacking a subset of methionyl and tryptophanyl residues whose oxidation mediates the dissociation of the native homotetramer into conformationally locked dimers.