Electrospray mass spectrometric analysis of the domains of a large enzyme: observation of the occupied cobalamin-binding domain and redefinition of the carboxyl terminus of methionine synthase.
Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the methylation of homocysteine to form methionine, using methyltetrahydrofolate as the primary methyl donor. We have used electrospray mass spectrometry as a powerful tool for characterizing separable fragments obtained by proteolysis of this monomeric 136.1-kDa enzyme. A central 28.0-kDa domain, reported to bind the cobalamin, has been purified to homogeneity in 30% yield. We were able to detect the domain with bound cobalamin by electrospray mass spectrometry at neutral pH. Mass analysis of a 37.2-kDa carboxyl-terminal domain was grossly inconsistent with either of the two amino acid sequences from previously published DNA sequences. We then used electrospray mass spectrometry to analyze peptides generated by a lysyl endoproteolytic digest of a C-terminal fragment, and we have constructed a peptide map that accounts for > 95% of the peptide mass derived from this domain. The correct translational end of this protein (27 residues downstream from the previously predicted ultimate residue) has been established, and sequence conflicts within the two published DNA sequences have been resolved (GenBank Accession Number J04975). Resequencing the DNA near the carboxyl terminus ruled out a frameshifted reading of the DNA and suggested that a cytosine had twice been incorrectly inserted late in the reading frame. The strategies reported here for sequence confirmation, localization of coenzyme-binding regions, and identification of chemically modified peptides within a large protein are potentially applicable to the characterization of many other proteins.
Drummond
J T
JT
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Loo
R R
RR
Matthews
R G
RG
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009609:Nitrous Oxide / Q000378:metabolism; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D014805:Vitamin B 12 / Q000378:metabolism*
D004269:DNA, Bacterial; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D009609:Nitrous Oxide; D014805:Vitamin B 12