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.
Lindquist
J A
JA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, USA.
Barofsky
E
E
McFadden
P N
PN
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000032:analysis / Q000737:chemistry; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D008745:Methylation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011497:Protein O-Methyltransferase / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D010455:Peptides; D001224:Aspartic Acid; D001216:Asparagine; D011497:Protein O-Methyltransferase; D014357:Trypsin; D003488:Cyanogen Bromide