Role of calcium ions in the structure and function of the di-isopropylfluorophosphatase from Loligo vulgaris.
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.
Hartleib
J
J
Institute of Biophysical Chemistry, Johann Wolfgang Goethe-University of Frankfurt/M., Marie-Curie-Strasse 9, 60439 Frankfurt/M., Germany.
Geschwindner
S
S
Scharff
E I
EI
Rüterjans
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002942:Circular Dichroism; D049832:Decapodiformes; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D044345:Phosphoric Triester Hydrolases; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013725:Terbium / Q000378:metabolism
D013725:Terbium; D004950:Esterases; D044345:Phosphoric Triester Hydrolases; C020061:diisopropyl-fluorophosphatase; D002118:Calcium