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 catalyze s the methylation of homocysteine to form methionine, using methyltetr ahydrofolate as the primary methyl donor. We have used electrospray ma ss spectrometry as a powerful tool for characterizing separable fragme nts obtained by proteolysis of this monomeric 136.1-kDa enzyme. A cent ral 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 b ound cobalamin by electrospray mass spectrometry at neutral pH. Mass a nalysis of a 37.2-kDa carboxyl-terminal domain was grossly inconsisten t with either of the two amino acid sequences from previously publishe d DNA sequences. We then used electrospray mass spectrometry to analyz e 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 translatio nal end of this protein (27 residues downstream from the previously pr edicted ultimate residue) has been established, and sequence conflicts within the two published DNA sequences have been resolved (GenBank Ac cession Number J04975). Resequencing the DNA near the carboxyl terminu s ruled out a frameshifted reading of the DNA and suggested that a cyt osine had twice been incorrectly inserted late in the reading frame. T he strategies reported here for sequence confirmation, localization of coenzyme-binding regions, and identification of chemically modified p eptides within a large protein are potentially applicable to the chara cterization of many other proteins.