Repair of Isopeptide Bonds by Protein Carboxymethyl Transferase: Seminal RNase as a Model System

Previous work has shown that in the peptide segment 62-76 of naturally deamidated a subunit of bovine seminal ribonuclease (BS-RNase) the a-carboxyl group of iso- Asp67 is selectively methylated by S-adenosy1methionine:protein carboxyl 0-methyltransferase. In the present study this reaction has been characterized, by using the tryptic segment 62-76 of the protein chain (peptide a16). The peptide is stoichiometrically methyl esterified with a KM of 6.17 pM and a Vmax of 19.56 nmol min-1 mg-1, and the product of demethylation has been identified as the cyclic succinimidyl derivative of iso-Asp67-Gly68. The cleavage of the succinimidyl ring yields two isomeric peptides containing an aspartyl residue (peptide a17) and an isoaspartyl residue (peptide a16). On the basis of these results conditions were defined in which repeated cycles of methylation-demethylation led to an effective conversion of peptide a16 into peptide a17, a process that can be interpreted as the repair of an altered isopeptide bond. When the methyl esterification reaction was studied on the native dimeric isoenzymes of seminal RNase and on catalytically active monomeric derivatives, including a stabilized alfa-type subunit, the results of these experiments showed that none of the protein forms were substrates for the methyltransferase. Only the unfolded alfa-type subunit was methylated to a stoichiometric extent. These results indicate that the repair of altered isopeptide bonds is chemically feasible in peptides but is hindered in the case of seminal RNase by its three-dimensional structure.