Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.

Abstract

The reaction of methylamine with .alpha.2-macroglobulin (.alpha.2M) results in the covalent modification of one gluamic residue per subunit as .gamma.-glutamylmethylamide .alpha.2M can undergo specific peptide autolysis involving the same reactive glutamic residue. During both reactions, a cysteinyl thiol is exposed and can be alkylated by iodoacetic acid. After .alpha.2M was modified with [14C]methylamine and iodo[2-3H]aetic acid, a tryptic peptide was isolated that contained both labels in the same ratio as in the original protein. From the chymotryptic digest of the tryptic peptide, a single radiolabeled peptide was isolated. The amino acid sequence of the chymotryptic peptide was the same as that previously reported to include .gamma.-glutamylmethylamide. This is circumstantial evidence for a thiolester between the cysteine and a glutamic acid located 3 residues away in the primary sequence. A reaction mechanism involving a pyroglutamyl intermediate derived from the thiolester is suggested to explain the autolysis. Kinetic analysis of the autolysis reaction is consistent with this intermediate and mechanism.