title: 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 × 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.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Mousumi
authors.initials: M
authors.lastname: Banerjee
authors.forename: Kallol
authors.initials: K
authors.lastname: Gupta
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Balaram
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Balaram
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D017015:Methanococcales / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D058767:Protein Unfolding; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D010446:Peptide Fragments; D014305:Triose-Phosphate Isomerase