Complexation of labile metal ions and effect on toxicity

Chelating agents that have been or may be employed for the treatment of lead, iron, and aluminum overload conditions are described. Although EDTA has been used successfully for treatment of acute lead poisoning, superior ligands for this purpose can be developed. The use of desferriferrioxamine B (DFB) for the treatment of iron overload has several disadvantages, and superior iron chelators are now available. Aluminum overload may also be treated with DFB, but many superior ligands, including chelators designed for iron, are now available for clinical tests.     

Iron-sulphur clusters with labile metal ions.

A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd III from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [formula: see text] The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]0 cluster for divalent ions lie in the sequence Cd greater than Zn much greater than Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S]1+ as well as for [3Fe-4S]0. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-mu 2-sulphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least one additional ligand which may be from solvent (H2O or OH-) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn(II), that may trigger conformational change.     

Isotope effect studies of the role of metal ions in isocitrate dehydrogenase.

Pig heart NADP+-dependent isocitrate dehydrogenase requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the carbon isotope effect is k12/k13 = 0.9989 +/- 0.0004 for the carboxyl carbon undergoing decarboxylation and hydrogen isotope effects are VmaxH/VmaxD = 1.09 +/- 0.04 and (Vmax/Km)H/(Vmax/Km)D = 0.76 +/- 0.12 with threo-D,L-[2-2H]isocitric acid. Deuterium isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/VD = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be two or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about tenfold lower in the presence of Ni2+ than in the presence of Mg2+. The carbon isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 +/- 0.0012 and the hydrogen isotope effects are VmaxH/VmaxD = 0.98 +/- 0.07 and (Vmax/Km)H/(Vmax/Km)D = 1.11 +/- 0.14. Thus, the rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. However, a more detailed analysis of the carbon isotope effects reveals that there is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl oxygen of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.     

The role of metal ions in the activation of arginase.

The role of metal ions as activators of arginase hydrolyzing arginine were studied. The metal ion is assumed to form a complex with arginine and to promote the enzymatic reaction. The activating ability of the metal ion appears to be governed by the chelating ability and/or the coordination numbers which determine whether the metal ion combines with the enzyme or the substrate (or both substrate and enzyme) and factors which influence the configuration of the resulting complexes.     

Catalysis of phosphoryl group transfer. The role of divalent metal ions in the hydrolysis of lactic acid O-phenyl phosphate and salicylic acid O-aryl phosphates.

The spontaneous hydrolyses of lactic acid O-phenyl phosphate (I) and, to a lesser extent, 3-hydroxybutyric acid O-phenyl phosphate (II) have been investigated and compared with similar intramolecular and bimolecular reactions. Compared to bimolecular nucleophilic reactions, the reactivity of II is similar to other systems involving the formation of a six-membered ring intermediate, which suggests that the electrostatic barrier to attack of an anionic nucleophile on a phosphate diester anion is fully present in II. The reactivity of I, as compared to that of II, would suggest that at least a partial overcoming of the electrostatic barrier takes place upon closer approimation of the two reacting centers. The Mn-2+-catalyzed hydrolysis of I exhibits saturation kinetics, consistent with the enhanced reactivity of the metal ion-substrate complex. The binding constant for this complex, determined from kinetics, is in good agreement with that obtained by electron spin resonance (ESR) titration. It is argued that the complex of Mn-2+ with II, as observed by pulsed Fourier transform nuclear magnetic resonance (NMR) techniques, is a precursor to the complex of catalytic significance. The hydrolysis of I as catalyzed by a variety of divalent metal ions suggests an optimal metal ion size. The spontaneous and metal ion catalyzed hydrolyses of salicyclic acid O-aryl phosphates (IIIa-d) proceed through cyclic acyl phosphate intermediates after expulsion of phenol. Product studies on the parent compound have failed to detect phenyl phosphate as a product in either the spontaneous or metal ion catalyzed process. The dependence of the second-order rate constant for the metal-catalyzed hydrolysis on leaving group pKa, beta-1-g, decreases significantly relative to beta-1-g for the spontaneous hydrolysis. From the collective data a specific interation of the metal ion with a pentacovalent intermediate is inferred in the rate-determining step for esters I and III. The probable consequences of these mechanistic postulates for phosphoryl transfer reactions in biological systems are discussed.     

The role of metal ions in RNA catalysis

Understanding the catalytic mechanisms of RNA enzymes remains an important and intriguing challenge - one that has grown in importance since the recent demonstration that the ribosome is a ribozyme. At first, it seemed that all RNA enzymes compensate for the limited chemical versatility of ribonucleotide functional groups by recruiting obligatory metal ion cofactors to carry out catalytic chemistry. Mechanistic studies of the large self-splicing and pre-tRNA-processing ribozymes continue to support this idea, yielding increasingly detailed views of RNA active sites as scaffolds for positioning catalytic metal ions. Re-evaluation of the methodologies used to distinguish catalytic and structural roles for metal ions, however, has challenged this notion in the case of the small self-cleaving RNAs. Recent studies of the small ribozymes blur the distinction between catalytic and structural roles for metal ions, and suggest that RNA nucleobases have a previously unrecognized capacity for mediating catalytic chemistry.     

The role of metal ions in the conformation of the four-way DNA junction.

Metal ions fold DNA junctions into a compact conformation that confers protection of all thymine bases to modification by osmium tetroxide. In the absence of the cation the arms of the junction are fully extended in an approximately square-planar configuration. Group IIa cations are effective in achieving a folded conformation of the junction at 80-100 microM, and there is an excellent agreement between the ionic concentrations that fold the junctions as deduced from gel electrophoretic experiments, and those that prevent osmium tetroxide reaction at the junction. Hexamminecobalt(III) achieves full folding at 2 microM, while spermine and spermidine are effective at 25 microM. Some transition metal ions such as Ni(II) may replace the group IIA cations. Monovalent ions of group IA are only partially effective in folding the junctions. Very much higher concentrations are necessary, gel electrophoretic mobilities suggest that a less symmetrical conformation is adopted and thymine bases at the junction remain reactive to osmium tetroxide. Charge-charge interactions at the centre of the junction are structurally extremely important. Substitution of junction phosphate groups by uncharged methyl phosphonates severely perturbs the structure of the junction. If just two phosphates are substituted, diametrically facing across the junction, the structure always folds in order to place the electrically neutral phosphate on the exchanging strands. We suggest that folding of the junction into the stacked X-structure generates electronegative clefts that can selectively bind metal ions, depending on the chemistry, size and charge of the ion. Moreover, occupation of these cavities is essential for junction folding, in order to reduce electrostatic repulsion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical-chemical studies on the role of the metal ions in concanavalin A.

Substitution of Co²⁺ for Mn²⁺ in concanavalin A generates characteristic circular dichroism and magnetic circular dichroism spectra which are strongly affected by the concentration of Ca²⁺. With three equivalents of Ca²⁺ per protomer of [(Co²⁺)Con A], no spectral effects of addition of α-methyl-d-glucopyranoside can be demonstrated. With one equivalent of Ca²⁺, however, α-methyl-d-glucopyranoside alters the circular dichroism and magnetic circular dichroism spectra in a manner identical to that produced by adding further equivalents of Ca²⁺. Under these same conditions the higher molecular weight carbohydrates, trehalose and melezitose, cause no spectral alterations in the regions investigated.The magnetic circular dichroism spectrum of [(Co²⁺)Con A] is characterized by a negative peak centered at 510 nm (θ/gauss = ?0.28 °) and a pronounced shoulder at 462 nm (θ/gauss = ? 0.16 °). Comparison of this spectrum to that of Co(H₂O)₆²⁺ indicates that the transition metal ion exhibits octahedral geometry in solution and maintains this geometry in its interaction with carbohydrate moieties.Circular dichroism experiments in the far ultraviolet region indicate a change in secondary (presumed β) structure upon interaction of Apo Con A with Mn²⁺ consistent with a more ordered arrangement. Unlike Mn²⁺, cobalt alone will not induce these secondary changes until Ca²⁺ is added. Kinetic analysis, using a mannan light scattering assay, indicates that [(Mn²⁺)Con A] and [(Co²⁺)Con A] will slowly recover cross-linking function in the absence of Ca²⁺, suggesting that the role of the metal in S₂ is to accelerate a conformational change leading to binding or effector function.Overall, the data are consistent with a suggestion by Cuatrecasas (1973) that α-methyl-d-glucopyranoside binds to a locus different from the membrane binding (or agglutination) site. Nevertheless, there are strong conformational interactions between these two sites, since α-methyl-d-glucopyranoside will elute Con A from membrane surfaces.     

The effect of metal ions on mitochondrial pyridine dinucleotide transhydrogenase

Bovine heart submitochondrial particle transhydrogenase is inhibited by cations in a concentration and pH-dependent manner, and non-energy-linked transhydrogenation is inhibited to a greater extent by metals than the energy-linked reaction. The inhibition of the enzyme by Mg²⁺ is competitive with the NADP substrate and non-competitive with the NAD substrate. Mg²⁺ stimulates inactivation of the enzyme by 5,5′-dithiobis(2-nitrobenzoic acid), and protects against thermal and proteolytic inactivation. This suggests that Mg²⁺ binding in the NADP site alters transhydrogenase to a more thermostable conformation, which is less susceptible to attack by trypsin and more reactive with 5,5′-dithiobis(2-nitrobenzoic acid). Other cation inhibitors mimic Mg²⁺ in these properties. The order of effectiveness of the inhibitors tested is La³⁺ > Mn²⁺ > Ca²⁺ ? Mg²⁺ > Sr²⁺ > Na⁺ ? K⁺. This order is described by the Irving-Williams order for the stability of metal-ligand complexes, suggesting that carboxylates or amines may comprise the inhibitory cation binding site.     

The effect of metal ions on the alkaline phosphatase of Rhizobium leguminosarum

The alkaline phosphatase (EC 3.1.3.1.) from Rhizobium leguminosarum WU235 has been purified. The enzyme is a non-specific phosphomonoesterase, has a molecular weight of 78,500 and a sub-unit molecular weight of 39,400. Magnesium and zinc ions are implicated in the structure of the enzyme; atomic absorption analysis gave 1.9 g-atoms Mg²⁺ and 1.9–5.1 g-atoms Zn²⁺ per mole of enzyme. In addition high concentrations of Mg²⁺ markedly stimulate the enzyme. The phosphatase is inhibited by Li⁺ and Na⁺ and stimulated by K⁺, Rb⁺ and Cs⁺, which suggests that the enzyme is K⁺ activated.     

The effect of metal ions on the electrochemistry of the antitumor antibiotic streptonigrin

The effect of transition metal ions on the electrochemistry of 6-methoxy-5,8-quinolinedione (L1), 7-amino-6-methoxy-5,8-quinolinedione (L2) and the antitumor antibiotic streptonigrin (SN) was studied. In 10% methanol/water, the one-electron reduction of quinones L1 and L2 to the corresponding semiquinones is shifted to more positive potentials upon addition of one equivalent of Zn(II), Ni(II), Co(II) or Cd(II) and is consistent with formation of a 1:1 complex involving the quinone(N) and adjacent quinone(O). Similar results are observed for Cu(II) and Mn(II), but the redox chemistry is also complicated by metal-based redox chemistry. The addition of further equivalents of M(II) results in a number of different coordination and electrochemical processes including formation of 1:1 and 2:1 complexes of the quinone, semiquinone and dianion. Under similar conditions, the 1:1 SN 2,2'-bipyridyl metal complex undergoes a reversible one-electron reduction to the semiquinone. The redox potential of the quinone in SN was shifted positive in the presence of the metal ions, but both the magnitude of the shift, and the relative influence of the metals was different to ligands L1 and L2. The changes in redox chemistry of SN compared with L1 and L2 are consistent with the formation of the 2,2-bipyridyl complexes in which there is weaker coordination to the quinone(O) in ring A of SN. These results suggest that in vivo, metal ions such as Zn(II), Cu(II) and Mn(II) facilitate the initial reduction of streptonigrin to the semiquinone by capturing the semiquinone after SN is reduced by biological reductants.     

Complexing of 3d transition metal ions with 9-substituted purines. IV. The effect of base-stacking on the basicity and complexing-ability

A number of 9-methylpurines were equilibrated between carbon tetrachloride or chloroform and aqueous solutions containing either nickel(II) perchlorate or N⁶,N⁶-dimethyladenosine. The equilibrium constants for the complex formation with nickel(II) ion and association with N⁶,N⁶-dimethyladenosine were calculated on the basis of the distribution data. The results, together with those obtained in the presence of both nickel(II) perchlorate and N⁶,N⁶-dimethyladenosine, are interpreted to indicate that stacking-association with the latter compound reduces the complexing-ability of 9-methylpurines. The protonation of guanosine in the presence of caffeine was examined potentiostatically and its association with caffeine studied by phase-solubility measurements. Association with caffeine was shown to lower the basicity of guanosine.