An equilibrium model of the metastable binding sites of alpha 2-macroglobulin and complement proteins C3 and C4.

Abstract

Two cyclic structures, the 15-membered thiolactone A and the 5-membered lactam P, have been proposed for the metastable binding sites of the serum proteins C3, C4, and alpha 2-macroglobulin. Neither structure alone adequately explains two unusual reactions of these sites, namely, covalent attachment to nucleophiles with liberation of a thiol group and spontaneous hydrolysis (autolysis) of an internal peptide bond. The metastable binding sites of these proteins were modeled with the 15-membered thiolactone 1 (Khan, S. A., and Erickson, B. W. (1981) J. Am. Chem. Soc. 103, 7374-7376) and the isomeric 5-membered lactam 2, which contains an internal pyroglutamyl (Glp) residue. Under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C), thiolactone 1 and lactam 2 exist in dynamic equilibrium. Since the molar ratio of 2/1 is 11:1 at equilibrium, lactam 2 is 15 kcal/mol more stable than thiolactone 1. The activation energy for isomerization of 1 into 2 is 18 kcal/mol, which is about 5 kcal/mol lower than that for hydrolysis of the acyclic thiolester N,S-diacetyl-L-cysteine methylamide. Part of the chemistry of the metastable binding sites can be explained by an analogous equilibrium between protein structures A and B. Lactam B may be a key intermediate in the biosynthesis of thiolactone A. Under denaturating conditions, thiolactone A could either bind covalently to a receptive surface or isomerize into lactam B, which could undergo spontaneous hydrolysis of the Glu-Glp peptide bond.