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 providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion-spray) MS for the biochemical characterization of recombinant human catechol O-methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352 +/- 2 Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25775 +/- 4 Da. To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide 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 of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5 Da) is in good agreement with our experimental result. The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S-adenosyl-L-methionine (AdoMet) and MgCl2, was also studied. 5-Iodoacetamido fluorescein (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 tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5-IAF. Incorporation of 5-IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.
Vilbois
F
F
Pharma Division, New Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Caspers
P
P
da Prada
M
M
Lang
G
G
Karrer
C
C
Lahm
H W
HW
Cesura
A M
AM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002394:Catechol O-Methyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D003545:Cysteine; D004926:Escherichia coli / Q000378:metabolism*; D005452:Fluoresceins / Q000494:pharmacology; D016368:Frameshift Mutation; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D011392:Proline; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014357:Trypsin
D003062:Codon; D005452:Fluoresceins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C015296:5-iodoacetamidofluorescein; D012436:S-Adenosylmethionine; D011392:Proline; D002394:Catechol O-Methyltransferase; D014357:Trypsin; D003545:Cysteine