title: Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I--kinetic parameters, autolysis and thermal stability.
abstract: Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k(cat)/K(m)) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 degrees C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.
authors.affiliation: Department of Biochemistry, Science Institute University of Iceland, Dunhaga 3, 107 Reykjavík, Iceland.
authors.forename: Bjarki
authors.initials: B
authors.lastname: Stefansson
authors.forename: Linda
authors.initials: L
authors.lastname: Helgadóttir
authors.forename: Sigridur
authors.initials: S
authors.lastname: Olafsdottir
authors.forename: Agústa
authors.initials: A
authors.lastname: Gudmundsdottir
authors.forename: Jón B
authors.initials: JB
authors.lastname: Bjarnason
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003080:Cold Temperature; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D048389:Gadus morhua / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D014357:Trypsin