Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues.

D W Aswad

doi:10.1016/s0021-9258(18)90570-3

Abstract

The enzymatic methylation of porcine adrenocorticotropin (ACTH) in both its native form and a form which is deamidated at asparagine 25 has been compared using purified protein carboxyl methyltransferase from bovine brain. Incubation of deamidated ACTH with high concentrations of methyltransferase resulted in near stoichiometric levels of methyl incorporation (78 mol %), while the methylation of native ACTH was highly substoichiometric (3-12 mol %). The Km and Vmax for deamidated ACTH were 1.9 microM and 11,200 pmol/min/mg, respectively, making this peptide the most specific substrate known for the mammalian methyltransferase. Deamidation of asparagine 25 leads to the formation of an atypical isopeptide bond in which the resulting aspartyl residue is linked to the adjacent glycine 26 via its side-chain beta-carboxyl group rather than the usual alpha-carboxyl linkage (Gráf, L., Bajusz, S., Patthy A., Barát, E., and Cseh, G. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 415-418; Bornstein, P., and Balian, G. (1977) Methods Enzymol. 47, 132-145). A synthetic isopeptide (beta-linked) analog of deamidated ACTH serves as a highly effective substrate for the methyltransferase, but the corresponding normal (alpha-linked) peptide does not, indicating that this enzyme selectively recognizes the alpha-carboxyl group of atypical beta-linked L-aspartyl residues (see also accompanying paper (Murray, E.D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732]. Methylation of atypical beta-linked L-aspartyl residues resulting from deamidation can account for previous observations that in vitro protein carboxyl methylation in mammalian systems almost always occurs with a low stoichiometry and that these protein methyl esters are considerably less stable than most chemically formed protein methyl esters.

Highlights

(78 mol %), while the methylation of native ACTH was highly substoichiometric (3-12 mol %)
The corresponding normal (a-linked) peptide does not, in- protein methyl esters formed by the mammalian enzyme are dicating that thisenzyme selectively recognizesthe a- considerably less stable at neutral and alkaline pH than are carboxyl groupof atypical &linked L-aspartyl residuesthe bacterialmethylated chemoreceptors or the eucaryotic (see accompanyingpaper (Murray,E.D., Jr., and phosphoproteins [3, 29,30,31]
Methylation of atypical &linked L-aspartyl residues resulting from deamidation can account for previous observations that in vitro protein carboxyl methylation in mammalian systems almostalways occurs with a low stoichiometry and that these protein methesy-l ters areconsiderably less stable thanmost chemically formed proteinmethyl esters

Summary

RESULTS

Substoichwmetric Nature of Methyl-accepting Sites in ACTH-Kim and Li [34] have previously reported that ovine ACTH can be enzymatically carboxyl methylated with a relatively high stoichiometry (0.30 mol of CH3/mol of peptide). The exactratio of the two of deamidated ACTH yielded an apparentK, of 1.9 p~ and products has never been determined, the more acidic nature a Vmaxof 11,200 pmol/min/mg (Fig. 7) These favorable ki- of the a-carbon predicts that the @-linkedpeptide should be netic constants,together with the high stoichiometry of meth- the more abundant product [38]. As pointed out by Riniker abundant form of the enzyme which has a more acidic iso- et al [43], early attempts at sequencing ACTH by Edman electric point than thetype Iisozyme [26] These experiments degradation failed at position 25 due presumably to a large indicate that the kinetic constants for the methylation of degree of deamidation during previous sequencing cycles. Thesepeptides were kindly provided to us by Drs David Murray and Steven Clarke of the University of California, Los Angeles, who synthesized them in the course of their studies on the substrate specificity of protein carboxyl methyltransferaseinhuman erythrocytes [57]

CYCLIC IMIDE INTERMEDIATE

DISCUSSION

Hexapeptide assayb

Thereis considerableevidencealready intheliterature