Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate. Detection of the reaction products by fast-atom-bombardment mass spectrometry.
Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-602], while very little is known concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11,503-11,511]; (2) the isoaspartyl residue is not recognized unless its alpha-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methyl-esterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.
Galletti
P
P
Istituto di Biochimica della Macromolecole, I Facoltà di Medicina e Chirurgia, Università di Napoli.
Ingrosso
D
D
Manna
C
C
Sica
F
F
Capasso
S
S
Pucci
P
P
Marino
G
G
D002384:Catalysis; D004952:Esters / Q000378:metabolism; D013058:Mass Spectrometry; D008745:Methylation; D009842:Oligopeptides / Q000378:metabolism*; D011496:Protein Methyltransferases / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D013379:Substrate Specificity
D004952:Esters; D009842:Oligopeptides; D011496:Protein Methyltransferases; D011497:Protein O-Methyltransferase