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: Bjarki Stefansson, Linda Helgadóttir, Sigridur Olafsdottir, Agústa Gudmundsdottir, Jón B Bjarnason
• Journal: Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology (Comp Biochem Physiol B Biochem Mol Biol) Vol. 155 Issue 2 Pg. 186-94 (Feb 2010) ISSN: 1879-1107 [Electronic] England
• PMID: 19913635 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Enzyme Inhibitors
• Trypsin

Topics

• Adaptation, Physiological
• Animals
• Binding Sites
• Cattle
• Cold Temperature
• Enzyme Inhibitors (pharmacology)
• Enzyme Stability
• Gadus morhua (metabolism, physiology)
• Kinetics
• Spectrometry, Fluorescence
• Trypsin (chemistry, isolation & purification, metabolism)