Abstract
Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.
Highlights
Protein L-isoaspatrtyl methyltransferase (PIMT) enzyme appears to be distinct from recently reported transfers the methyl groupof S-adenosyl-L-methionine methyltransferase activities which modify the H-ras protein to free a-carboxyglroupsof atypical L-isoaspartylres- [13] and other putative guanine nucleotide-binding proteins idues in proteins
It is possible that other knownclass of methyltransferase including isoaspartate is present as a normal component of certain those which methylate the side chain y-carboxyl group proteins and that themethylation of these sites constitutes a of receptor proteins involved in bacterial chemotaxis. hitherto unknown regulatory or processing pathway
All peptides obtained from enzymatic and chemical cleavages of PIMT were analyzed by FAB mass spectrometry prior to Edman degradation
Summary
Protein L-isoaspatrtyl methyltransferase (PIMT) enzyme appears to be distinct from recently reported transfers the methyl groupof S-adenosyl-L-methionine methyltransferase activities which modify the H-ras protein to free a-carboxyglroupsof atypical L-isoaspartylres- [13] and other putative guanine nucleotide-binding proteins idues in proteins. All peptides were analyzed by FAB mass spectrometry which served to confirm the automated Edman data. Automated Edman degradation of eight Lys-C peptides, KC7, KC21, KC16, KC3, KC5, KC14, KC10, and KC9, provided the sequences shown in Tables 1 and 2. Mixture sequence analysis of peak 24 yielded CN24a and CN24b resultingin the sequences shown, which were confirmed by FAB mass spectrometry.
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