Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.
We provide here the first direct evidence that D-aspartyl residues in peptides are substrates for the L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). We do this by showing that D-aspartic acid beta-methyl ester can be isolated from carboxypeptidase Y digests of enzymatically methylated D-aspartyl-containing synthetic peptides. The specificity of this reaction is supported by the lack of methylation of L-aspartyl-containing peptides under similar conditions. Methylation of D-aspartyl residues in synthetic peptides was not observed previously because with Km values ranging from 2.5 to 4.8 mM, these peptides are recognized by the methyltransferase with 700-10,000-fold lower affinity than are their L-isoaspartyl-containing counterparts. The physiological significance of D-aspartyl methylation was investigated in two ways. First, analysis of in situ methylated human erythrocyte proteins showed that at least 22% of the methyl groups associated with the proteins ankyrin and band 4.1 are on D-aspartyl residues, suggesting that D-aspartyl methylation is an important function of the methyltransferase in vivo. Second, mathematical modeling of the protein aging and methylation reactions occurring in intact erythrocytes indicated that the accumulation of D-aspartyl residues can be reduced as much as 2-5-fold by the methyltransferase activity. Although this reduction is much less than that predicted for L-isoaspartyl residues, it may be significant in maintaining functional proteins throughout the 120-day life span of these cells.
Lowenson
J D
JD
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
Clarke
S
S
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D002268:Carboxypeptidases; D004905:Erythrocyte Aging; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000302:isolation & purification; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000097:blood*; D013379:Substrate Specificity
D009842:Oligopeptides; D010446:Peptide Fragments; D001224:Aspartic Acid; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase