Figure 1.
Reaction model including two metal (X) binding sites affecting the kinetics of zebrafish ADPRibase-Mn.
Model assumptions are as follows. (i) X can bind to the enzyme (E) at two independent sites with dissociation constants KA (site A) and KB (site B) to give complex EX and XE, respectively; binding to either site does not affect binding to the other. (ii) On site A, X acts as an essential activator, required for substrate binding. (iii) On site B, X behaves as a general modifier that can increase or decrease the reaction rate depending on other conditions, e.g. the particular substrate, by modifying KS (to K′S) and/or k to k′ (i.e. V to V′). (iv) Binding of X to free S is negligible. From this model, kinetic equation I was derived and fit to results from experiments in which the added metal was Mn2+ (Fig. 3). As discussed in the text, in these experiments it was assumed that E is an ADPRibase-Mn form with one metal ion (Fe) strongly bound in the dimetalic center. Site A corresponds to the other position of the dimetalic center, and site B would be located outside the active center.
Figure 2.
Hydrolysis of 2′,3′-cAMP by zebrafish wild type- and H97A-ADPRibase-Mn.
Enzyme reaction mixtures with 500 µM 2′,3′-cAMP and 5 mM MnCl2 were incubated for 50 min. Similar unit amounts of wild-type and mutant enzymes were used to facilitate comparison. Each trace is a differential chromatogram showing the difference between samples incubated with enzyme minus no-enzyme controls. The arrows mark the retention times of commercial standards of the indicated compounds. The insert boxes are 5-fold amplifications of the 2′-AMP peaks. The numbers correspond to percent 2′,3′-cAMP consumption or 3′-AMP and 2′-AMP formation (means±S.D. of 3 assays).
Table 1.
Substrate specificity of ADPRibase-Mn enzymes.
Figure 3.
Kinetic response of zebrafish ADPRibase-Mn to changes of Mn2+ concentration with different substrates.
Rates of substrate hydrolysis were measured in standard reaction mixtures except that Mn2+ concentration was varied as indicated. The curves show the best fits of equation I to data points. Error bars correspond to mean±S.D. (n = 3). The goodness of the fit is given for each adjustment in terms of R2.
Figure 4.
Comparative responses of ADPRibase-Mn enzymes to Mn2+ and Mg2+ as activators.
Rates of substrate hydrolysis were measured in standard reaction mixtures except that Mn2+ concentration was varied as indicated, or that Mg2+ substituted for Mn2+. Error bars correspond to mean±S.D. (n = 3).
Figure 5.
Inhibition of the Mn2+-dependent activity of zebrafish ADPRibase-Mn by Zn2+, Fe2+ and Fe3+.
Rates of substrate hydrolysis were measured in standard reaction mixtures except that Mn2+ concentration was fixed at 30 µM, and the indicated concentrations of an inhibitor cation was added in the form of chloride salts. Error bars correspond to mean±S.D. (n = 3).
Figure 6.
pH-activity profiles of zebrafish ADPRibase-Mn.
Rates of substrate hydrolysis were measured in standard reaction mixtures except that different pH buffers were used: Tris/acetate (pH 6.0–7.0), Tris-HCl (pH 7.5–9.5), glycine/NaOH (pH 9.8–10.8). The pH values shown in the plots were measured in the reaction mixtures. Error bars correspond to mean±S.D. (n = 3).
Figure 7.
Substrates docked to the crystal structure of zebrafish ADPRibase-Mn.
(A) General view with ADP-ribose docked to the active site like in the first individual pose shown in C. (B) Upper view of A showing (left) the full protein surface and (right) a view with part of the protein removed to allow vision of bound ADP-ribose. (C) Series of poses found for the different subtrates by docking. For ADP-ribose, CDP-ethanolamine and 2′,3′-cAMP, two poses with different orientations are shown. The histidine residue of the GNH[D/E] motif, His-97, is shown in every pose. Bronze spheres, metal ions of the dinuclear center; red sphere, bridging molecule of water, which is assumed to act as nucleophile in the ADPRibase-Mn reactions. The figures were prepared with the Visual Molecular Dynamics (VMD) program [50].
Figure 8.
Structural elements unique to the ADPRibase-Mn-like family within the MDP superfamily.
(A) Conservation of sequence and structure of prototypical zebrafish ADPRibase-Mn (PDB ID 2nxf) within the SCOP MDP superfamily. From the 44 structural homologues returned by a DALI search (Fig. S1), a group of 14 structures was selected (Table S1), including the closest structural homologue and members of the other 11 families of the superfamily. A structural alignment of 2nxf and these 14 homologues was generated with the VMD MultiSeq plugin [51], and it was used to color zebrafish ADPRibase-Mn by sequence or structure conservation (blue, conserved; red, not conserved). Bronze spheres, metal ions. (B) Topology diagram of zebrafish ADPRibase-Mn according to structural elements summarized in PDBSum (http://www.ebi.ac.uk/pdbsum; triangle, β strand; circle, α-helix, except 6, 8, 10 and 12 which are 310 helices). The two βαβαβ motives are shaded in different gray tones. Parts shaded in red are the unique elements of zebrafish ADPRibase-Mn delimiting the active center entrance. Bronze circles, metal ions; small arrows and names, amino acids coordinated to metals in the dinuclear center.