Reversible inactivation of alkaline phosphatase from Atlantic cod (Gadus morhua) in urea.
Alkaline phosphatase (AP) from Atlantic cod (Gadus morhua) is a zinc and magnesium containing homodimer that requires the oligomeric state for activity. Its kinetic properties are indicative of cold-adaptation. Here, the effect of urea on the structural stability was studied in order to correlate the activity with metal content, the microenvironment around tryptophan residues, and events at the subunit interface. At the lowest concentrations of urea, the first detected alteration in properties was an increase in the activity of the enzyme. This was followed by inactivation, and the release of half of the zinc content when the amount of urea reached levels of 2 M. Intrinsic tryptophan fluorescence and circular dichroism ellipticity changed in the range 2.5 to 8 M urea, signaling dimer dissociation, followed by one major monomer unfolding transition at 6-8 M urea as indicated by ANS fluorescence and KI fluorescence quenching. Gibbs free energy was estimated by the linear extrapolation method using a three-state model as 8.6 kcal/mol for dimer stability and 11.6 kcal/mol for monomer unfolding giving a total of 31.8 kcal/mol. Dimer association had a very small ionic contribution. Dimers were stable in relatively high concentration of urea, whereas the immediate vicinity around the active site was vulnerable to low concentrations of urea. Thus, inactivation did not coincide with dimer dissociation, suggesting that the active site is the most dynamic part of the molecule and closest related to cold-adaptation of its enzymatic activity.
Asgeirsson
Bjarni
B
Department of Biochemistry, Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland. bjarni@raunvis.hi.is
Guojónsdóttir
Katrín
K
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002942:Circular Dichroism; D019281:Dimerization; D005453:Fluorescence; D048389:Gadus morhua / Q000378:metabolism*; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D014508:Urea; D000469:Alkaline Phosphatase