α2-Macroglobulin-protease reactions: Relationship of covalent bond formation, methylamine reactivity, and specific proteolysis

Abstract

Experiments were performed to define the relation between covalent binding of enzymes to β 2-macroglobulin ( α 2M), the specific proteolysis of α 2M subunits to 85K fragments, and the reactivity of the methylamine site on α 2M. We studied the reaction of α 2M with native trypsin, anhydrotrypsin, and two active lysyl-blocked derivatives, methyl-trypsin and dimethylmaleyl-trypsin, the last with reversibly modified amino groups that can be regenerated at low pH. The results were: (1) All enzymes tested reacted with α 2M but only native trypsin formed covalent complexes (not dissociable by sodium dodecyl sulfate). Trypsin and the lysyl-blocked enzymes caused complete proteolysis of the α 2M subunits, in agreement with previous studies. (2) The dimethyl-maleyl-trypsin became covalently bound to α 2M only after removing the blocking groups of the bound enzyme, indicating that sequential proteolysis and covalent bond formation is possible. Under the conditions used for deblocking, there was no change in the covalent/noncovalent binding ratio of native trypsin, anhydrotrypsin, or the other lysyl-blocked derivative, methyl-trypsin. (3) Native trypsin or anhydrotrypsin displaced methyl- or dimethylmaleyl-trypsin from their α 2M complexes but the newly bound enzymes with free amino groups did not form covalent bonds indicating that enzymes must remain in association with the inhibitor for the bond to form. (4) Methylamine reacts with noncovalent α 2M complexes but not with covalent complexes. (5) Methylamine-treated α 2M can still form complexes with trypsin but at a drastically reduced rate and only noncovalent complexes are formed. In summary, sequential proteolysis and covalent bond formation is possible under certain conditions, and there is a strong correlation between covalent binding and loss of methylamine reactivity. The latter observation is suggestive evidence for the identity of the covalent binding site of α 2M and the putative thiol ester of the methylamine site. The enzyme lysyl amino groups, are likewise possible candidates for attacking nucleophile at that site.