Enzymic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover

Abstract

At pH 7.4, 37.degree. C, bovine brain protein carboxymethyltransferase transiently methylates deamidated ACTH. The methylation occurs at the .alpha.-carboxyl group of an atypical .beta.-carboxyl-linked isoaspartyl residue (position 25). The immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, the inability of the demethylated product to be remethylated, the charge of this product, and its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either .alpha.- or .beta.-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. Protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking or some other as yet undiscovered alteration of protein structure.