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

The triosephosphate isomerase from the hyperthermophilic organism Methanocaldococcus jannaschii ( Mj TIM) 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. Copyright © 2011 John Wiley & Sons, Ltd.