Mass spectrometric identification of an intramolecular disulfide bond in thermally inactivated triosephosphate isomerase from a thermophilic organism Methanocaldococcus jannaschii

Rapid Commun Mass Spectrom. 2011 Jul 30;25(14):1915-23. doi: 10.1002/rcm.5058.


The triosephosphate isomerase from the hyperthermophilic organism Methanocaldococcus jannaschii (MjTIM) is a tetrameric enzyme, with a monomer molecular mass of 23245 Da. The kinetic parameters, the k(cat) and the K(m) values, of the enzyme, examined at 25 °C and 50 °C, are 4.18 × 10(4) min(-1) and 3.26 × 10(5) min(-1) , and 0.33 and 0.86 mM(-1) min(-1) , respectively. Although the circular dichroism and fluorescence emission spectra of the protein remain unchanged up to 95 °C, suggesting that the secondary and tertiary structures are not lost even at this extreme temperature, surprisingly, incubation of this thermophilic enzyme at elevated temperature (65-85 °C) results in time-dependent inactivation, with almost complete loss of activity after 3 h at 75 °C. High-resolution electrospray ionization mass spectrometry (ESI-MS) reveals the monomeric mass of the heated sample to be 23243 Da. The 2 Da difference between native and heated samples suggests a probable formation of a disulfide bridge between proximal cysteine thiol groups. Liquid chromatography (LC)/ESI-MS/MS analysis of tryptic digests in the heated samples permits identification of a pentapeptide (DCGCK, residues 80-84) in which a disulfide bond formation between Cys81 and Cys83 was established through the collision-induced dissociation (CID) fragmentation of the intact disulfide-bonded molecule, yielding characteristic fragmentation patterns with key neutral losses. Neither residue is directly involved in the catalytic activity. Inspection of the three-dimensional structure suggests that subtle conformation effects transmitted through a network of hydrogen bonds to the active site residue Lys8 may be responsible for the loss of catalytic activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Circular Dichroism
  • Disulfides / chemistry*
  • Disulfides / metabolism
  • Hot Temperature
  • Methanococcales / enzymology*
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Unfolding
  • Sequence Analysis, Protein
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Tandem Mass Spectrometry
  • Triose-Phosphate Isomerase / chemistry*
  • Triose-Phosphate Isomerase / metabolism


  • Bacterial Proteins
  • Disulfides
  • Peptide Fragments
  • Triose-Phosphate Isomerase