Open AccessReactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.
Author(s) -
James B. Howard
Publication year - 1981
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.78.4.2235
Subject(s) - peptide , methylamine , chemistry , autolysis (biology) , alpha 2 macroglobulin , iodoacetic acid , cysteine , iodoacetamide , peptide sequence , biochemistry , stereochemistry , residue (chemistry) , amino acid , macroglobulin , enzyme , gene
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.