Conformation and stability of elastase from Atlantic cod, Gadus morhua.
Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.
Jayaraman
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G
Centre for Protein Engineering and Biomedical Research, The Voluntary Health Services, Adyar, Chennai 600 113, India. raman291068@yahoo.com
Srimathi
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S
Bjarnason
J B
JB
D000222:Adaptation, Physiological; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003080:Cold Temperature; D004795:Enzyme Stability; D048389:Gadus morhua; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019216:Metals, Heavy / Q000494:pharmacology; D010196:Pancreatic Elastase / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature
D019216:Metals, Heavy; D010196:Pancreatic Elastase; D019791:Guanidine; D002118:Calcium