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

Abstract

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.

Structural and Mutational Analyses of Drp35 from Staphylococcus aureus

“…These proteins have the common enzymatic feature of calcium dependence (9,35). Two calcium ions were observed in the Drp35-Ca 2ϩ complex, one of which (Ca1 in Fig.…”

“…These two eukaryotic proteins are classified as phosphotriesterases (EC 3.1.8) and possess a common biological activity (i.e. calcium-dependent hydrolysis activity) (9,35). Similarly, Drp35 can hydrolyze lactones in a calcium-dependent manner and is a functional counterpart of PON (i.e.…”

Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion

“…500 µM) was prepared by dissolution in buffer (Chelex 100-treated 10 mM Hepes/NaOH, pH7.5, and 10 mM NaCl). The concentration of the Quin2 stock solution was determined by using the molar absorption coefficient, ε 240 , of 4.2 × 10 4 M −1 · cm −1 , following a 1:100 dilution of stock solution in the presence of 10 mM CaCl 2 [42]. LACD (lowaffinity-Ca 2+ -ion-depleted) enzymes used for the Quin2 titration experiments were prepared by dialysis as described above.…”

“…Background fluorescence spectra of water and buffer were identical and showed no calcium contamination. Calcium-dissociation constants (K dCa 2+) for proteins can be calculated using several methods [42,43], assuming that the increase in fluorescence intensity is proportional to the concentration of the Quin2-Ca 2+ complex [44]. In the present study, in the presence of the calcium-binding protein, the apparent dissociation constant of the Quin2-Ca 2+ complex, K d(QCa) , was calculated by fitting experimental data to the following equation [42]:…”

“…Quin2 has been utilized to probe high-affinity calcium-binding given its own high affinity (K Q = 1.9 × 10 8 M −1 ) for Ca 2+ ions and the changes in its fluorescent properties after calcium binding [42]. Quin2 fluoresces with an emission maximum at 492 nm, which is far away from that of proteins, and its fluorescence is enhanced approx.…”