Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.
Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.
Asgeirsson
B
B
Department of Chemistry, Science Institute, University of Iceland, Reykjavik, Iceland. bjarni@raunvis.hi.is
Hauksson
J B
JB
Gunnarsson
G H
GH
D000222:Adaptation, Physiological; D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003080:Cold Temperature; D019281:Dimerization; D004795:Enzyme Stability / Q000187:drug effects; D005399:Fishes; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D010710:Phosphates / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D013816:Thermodynamics
D008024:Ligands; D010710:Phosphates; D000469:Alkaline Phosphatase; D019791:Guanidine