Dissociation and unfolding of coldâactive alkaline phosphatase from Atlantic cod in the presence of guanidinium chloride
2000
Ãsgeirsson, Bjarni | Hauksson, Jónas B. | Gunnarsson, Gudmundur H.
Coldâadaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a coldâactive 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, sizeâexclusion 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 threeâstate model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a twoâstate model). The ΔG for dimer dissociation of cod AP was 8.3âkcal·mol−1, and the monomer stabilization free energy was 2.2âkcal·mol−1, giving a total of 12.7âkcal·mol−1, whereas the total free energy of calf intestinal AP was 17.3âkcal·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 rateâlimiting 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.
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