Determination of two sites of automethylation in bovine erythrocyte protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase.

Protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed the alpha PCM fraction [Lindquist and McFadden (1994), J. Protein Chem. 13, 23-30]. The altered aspartyl sites serving as methyl acceptors in alpha PCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [3H]automethylated enzyme that contain a [3H]methyl ester. Methylation was positively identified at positions Asn188 and Asp217 in the enzyme sequence, a consequence of the spontaneous alteration of these sites to L-isoaspartyl or D-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows that alpha PCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.