MASS-SPECTROMETRIC ANALYSIS OF HUMAN SOLUBLE CATECHOL O-METHYLTRANSFERASE EXPRESSED IN ESCHERICHIA-COLI - IDENTIFICATION OF A PRODUCT OF RIBOSOMAL FRAMESHIFTING AND OF REACTIVE CYSTEINES INVOLVED IN S-ADENOSYL-L-METHIONINE BINDING

Technological advances in the field of mass spectrometry (MS) are prov iding powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted elec trospray (ion-spray) MS for the biochemical characterization of recomb inant human catechol O-methyltransferase (rhCOMT). hCOMT could be expr essed in Escherichia coli in large quantities but in two forms of diff erent size, both enzymically active. Electrospray MS analysis showed t hat the smaller rhCOMT protein had a molecular mass of 24352 +/- 2Da, corresponding to the calculated value for native hCOMT (without the in itiating methionine), whereas that mass of the larger protein was of 2 5775 +/- 4Da. To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced a nalysed by electrospray MS. Neither protein apparently contained disul fide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C-terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C-terminal extension resulted from ribosomal frameshift at the codon o f the last proline (CCC, rare codon in prokaryotes). In fact, rightwar d frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of th is protein (25773.5Da) is in good agreement with our experimental resu lt. The differential reactivity of the cysteine residues of the correc t rhCOMT enzyme, in the presence and in the absence of S-adenosyl-L-me thionine (AdoMet) and MgCl2, was also studied. 5-Iodoacetamido fluores cein (5-IAF) was used as thiol-modifying reagent. Under the conditions used, 5-IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl2 partially protected it from inactivation. The 5-IAF-labeled try ptic peptides were separated by HPLC and then submitted to electrospra y MS and tandem MS. Several cysteine residues appeared to be readily a vailable to chemical modification by 5-IAE Incorporation of 5-IAF occu rred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet an d MgCl2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or nea r the binding site of AdoMet.