Enzymatic methyl esterification of a deamidated form of mouse epidermal growth factor.
The enzyme S-adenosylmethionine:protein carboxyl-O-methyl-transferase, type II (EC 2.1.1.77; PCMT) from eukaryotes methyl esterifies peptides containing isoAsp residues, which can arise from spontaneous deamidation of labile Asn residues. We report here a study on in vitro methyl esterification of mouse EGF by bovine brain PCMT. This peptide contains two Asn in the sequences Asn1-Ser2 and Asn16-Gly17. It is known from the literature that the presence of a small residue on the carboxyl side of asparaginyl makes this residue susceptible to deamidation through the spontaneous formation of a succinimide intermediate. Therefore EGF was incubated under deamidating conditions (pH 9.0, 37 degrees for 48 h) and the extent of deamidation monitored by enzymatically measuring the NH3 produced during the alkali treatment: a release of 0.80 mol NH3/mol EGF was calculated. The alkali-treated EGF, analyzed by anion-exchange chromatography, shows two major components identified as native EGF (nEGF) and its deamidated form (dEGF). When incubated in the presence of purified PCMT neither nEGF nor dEGF showed any methyl accepting capability. Since it is known that the three-dimensional structure of a protein may hinder the methyl esterification of a potential ethyl accepting site, dEGF was unfolded by reducing and alkylating the intrachain disulfide bridges. Only a slight increase in the methyl accepting capability could be observed. Conversely, when EGF was deamidated after its unfolding, the resulting protein was stoichiometrically methylated by PCMT, presumably at level of isoAsp16. Our findings strongly suggest that the three-dimensional structure of a protein is a major specificity determinant for both deamidation and methyl esterification processes.
Galletti
P
P
Department of Biochemistry of Macromolecules, 1st Medical School, University of Naples, Italy.
Iardino
P
P
Ingrosso
D
D
Manna
C
C
Zappia
V
V
D000478:Alkylation; D000577:Amides / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D002853:Chromatography, Liquid; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004951:Esterification; D051379:Mice; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000378:metabolism*
D000577:Amides; D000596:Amino Acids; D004815:Epidermal Growth Factor; D000641:Ammonia; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase