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

• Published in: Biochemical journal Vol. 353; no. Pt 3; pp. 579 - 589
• Main Authors: Hartleib, J, Geschwindner, S, Scharff, E I, Rüterjans, H
• Format: Journal Article
• Language: English
• Published: England 01.02.2001
• Subjects: Animals; Calcium - metabolism; Calcium - physiology; Circular Dichroism; Decapodiformes; Electrophoresis, Polyacrylamide Gel; Esterases - chemistry; Esterases - metabolism; Kinetics; Phosphoric Triester Hydrolases; Protein Binding; Protein Conformation; Spectrometry, Fluorescence; Structure-Activity Relationship; Terbium - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/0264-6021:3530579

Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca(2+)-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 840 nM in the absence and the presence of 150 mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3 nM. Stopped-flow experiments have shown that the dissociation of bound Ca(2+) occurs in two phases, with rates of approx. 1.1 and 0.026 s(-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 Ca(2+) 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 Ca(2+) from DFPase. The Ca(2+) 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 Ca(2+) dissociation and the denaturation results we conclude that the higher-affinity Ca(2+) site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.