Role of calcium ions in the structure and function of thedi-isopropylfluorophosphatase from Loligo vulgaris

Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca2+-binding sites, which are required for catalytic activity and stability. Incubation with chelating agents results in the irreversible inactivation of DFPase. From titrations with Quin 2 [2-({2-[bis(carboxymethyl)amino]-5-methylphenoxy}-methyl)-6-methoxy-8-[bis(carboxymethyl)-amino]quinoline], a lower-affinity site with dissociation constants of 21 and 840nM in the absence and the presence of 150mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3nM. Stopped-flow experiments have shown that the dissociation of bound Ca2+ occurs in two phases, with rates of approx. 1.1 and 0.026s-1 corresponding to the dissociation from the low-affinity and high-affinity sites respectively. Dissociation rates depend strongly on temperature but not on ionic strength, indicating that Ca2+ dissociation is connected with conformational changes. Limited proteolysis, CD spectroscopy, dynamic light scattering and the binding of 8-anilino-1-naphthalenesulphonic acid have been combined to give a detailed picture of the conformational changes induced on the removal of Ca2+ from DFPase. The Ca2+ dissociation is shown to result in a primary, at least partly reversible, step characterized by a large decrease in DFPase activity and some changes in enzyme structure and shape. This step is followed by an irreversible denaturation and aggregation of the apo-enzyme. From the temperature dependence of Ca2+ dissociation and the denaturation results we conclude that the higher-affinity Ca2+ site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.