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

Abstract

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 4.18×10⁴min^-1 and 3.26×10⁵min^-1, and 0.33 and 0.86mM^-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.