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 glutamic residue per subunit as gamma-glutamylmethylamide [Swenson, R. & Howard, J. B. (1979) Proc. Natl. Acad. Sci. USA 76, 4313--4316]. Furthermore, alpha 2M can undergo specific peptide autolysis involving the same reactive glutamic residue [Howard, J. B., Vermeulen, M. & Swenson, R. (1980) J. Biol Chem. 255, 3820--3823]. 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]acetic 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 three 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.