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. T his paper describes studies on the conformational stability of a cold-activ e alkaline phosphatase (AP) from Atlantic cod, with the aim of understandin g more clearly its structural stability in terms of subunit dissociation an d unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography a nd enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction co uld be made between dissociation of dimers into monomers and subsequent unf olding of monomers (fits a three-state model). In contrast, dimer dissociat ion of calf AP coincided with the monophasic unfolding curve observed by tr yptophan fluorescence (fits a two-state model). The Delta G for dimer disso ciation 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 t he total free energy of calf intestinal AP was 17.3 kcal.mol(-1). Thus, dim er formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting di mer 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 di mer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.