Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation.

To map out the heavy metal binding sites of iduronic acid containing oligosaccharides isolated from human kidneys, we studied Zn(II) binding by nuclear magnetic resonance (NMR) and molecular modeling to two disaccharides isolated after nitrous acid depolymerization of heparin and two synthetic disaccharides representative of the heparin structure, namely, IdopA2S (alpha 1,4)AnManOH, 1 alpha, IdopA2S (alpha 1,4)AnManOH6S, 1b, IdopA2S-(alpha 1,4)GlcNS alpha Me, 2a, and IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b (see previous article in this series). A conformational analysis of the metal free and metal bound solutions was made by comparing calculated [(NOE)]s, [T1]s, and [J]s to experimental values. The 1C4, 4C1, and 2S0 conformations of the L-idopyranosiduronate ring and the 4E and 4T3 of the anhydro-D-mannitol ring are evaluated as are rotations about the C5-C6 hydroxymethylene of the AnManOH(6S) or GlcNS (6S) residues. The NOE between IdopA2S H1 and H3 and the known NOE between H2 and H5, as well as the T1 of IdopA2S H3, are introduced as NMR observables sensitive to the IdopA2S ring conformation. Similarly, a NOE between IdopA2S H5 and AnManOH(6S) or GlcNS(6S) H3 was observed that directly restricts the allowed interglycosidic conformational space. For all disaccharides, the Zn(II) bound spectral data are consistent with models in which these motions are partially "frozen" such that the 1C4 conformation of the IdopA2S is stabilized along with the 4T3 conformation of the AnManOH(6S) ring. The interglycosidic conformation is also stabilized in one of two minima. Electrostatic potential energy calculations gave the best overall agreement with experiment and suggest metal binding conformations with the carboxylate and ring oxygen of the IdopA2S residues (1C4 conformation) and either O3 of the GlcNS(6S) residues or the sulfate oxygens of the 6-sulphate for 2b providing additional chelating sites. These chelation models concur with the observation of marked 13C and 1H NMR chemical shifts for the IdopA2S resonances and of GlcNS H3 for 2 alpha and GlcNS6S C6 for 2b. This study of model compounds implicates the IdopA2S(alpha 1,4)GlcNS6S group as part of the heavy metal binding site in biologically important acidic oligosaccharides such as heparin.     

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.     

Conformation of complexes of thiamin pyrophosphate with divalent cations as studied by nuclear magnetic resonance spectroscopy.

The binding of Ni-2+ and Mn-2+ to thiamin phosphate and thiamin pyrophosphate (thiamin-PP) has been compared with the binding of these ions to oxythiamin phosphate and oxythiamin pyrophosphate, analogues of thiamin in which the C-4 amino group has been replaced by an -OH group. The replacement of the NH2 group results in reduced basicity of N-1 of the pyrimidine ring of oxythiamine derivatives. The effects of pD, ligand concentration, and temperature on the binding of metal ions to N-1 have been studied by observing the metal ion-induced shifting and broadening of the C-6-H signal of these compounds. The results indicate the following: (a) the metal ion is held near N-1, resulting in a "folded" conformation, because of a favorable bonding interaction between N-1 and the metal ion rather than for general conformational reasons alone; and (b) the amount of "folded" conformation present in the different pyrophosphate complexes at neutral pH follows the order: Ni-2+-thiamin-PP greater than Mn-2+-thiamin-PP greater than Mn-2+-oxythiamin-PP and Ni-2+-oxythiamin-PP It is concluded that the strength of the metal ion-pyrimidine interaction in the "folded" conformation depends strongly both on the coordination affinity of the metal ion and on the basicity of N-1. Since the interaction of the phosphate-bound metal ion with the pyrimidine ring in the Mg-2+-thiamin-PP complex is probably weaker than the corresponding interaction in the Mn-2+-thiamin-PP complex, these results predict that the Mg-2+-thiamin-PP complex in solution, at neutral pH, exists predominantly in an "unfolded" conformation.     

Metal ion enhanced binding of AMD3100 to Asp262 in the CXCR4 receptor

The affinity of AMD3100, a symmetrical nonpeptide antagonist composed of two 1,4,8,11-tetraazacyclotetradecane (cyclam) rings connected through a 1,4-dimethylene(phenylene) linker to the CXCR4 chemokine receptor was increased 7, 36, and 50-fold, respectively, by incorporation of the following: Cu(2+), Zn(2+), or Ni(2+) into the cyclam rings of the compound. The rank order of the transition metal ions correlated with the calculated binding energy between free acetate and the metal ions coordinated in a cyclam ring. Construction of AMD3100 substituted with only a single Cu(2+) or Ni(2+) ion demonstrated that the increase in binding affinity of the metal ion substituted bicyclam is achieved through an enhanced interaction of just one of the ring systems. Mutational analysis of potential metal ion binding residues in the main ligand binding crevice of the CXCR4 receptor showed that although binding of the bicyclam is dependent on both Asp(171) and Asp(262), the enhancing effect of the metal ion was selectively eliminated by substitution of Asp(262) located at the extracellular end of TM-VI. It is concluded that the increased binding affinity of the metal ion substituted AMD3100 is obtained through enhanced interaction of one of the cyclam ring systems with the carboxylate group of Asp(262). It is suggested that this occurs through a strong concomitant interaction of one of the oxygen's directly with the metal ion and the other oxygen to one of the nitrogens of the cyclam ring through a hydrogen bond.     

Independent and mutual interactions of copper(II) and zinc(II) ions with phytic acid

The interactions of phytic acid with Cu(II) and Zn(II) ions were examined as functions of metal ion concentrations and pH. Cu(II) ion-selective potentiometric and electron spin resonance (ESR) experiments provide strong evidence for the binding of Cu(II) ions to the phytic acid molecule at low pH (2.4–3.4) values. The relative stabilities of the copper and zinc phytates at low pH values were found to be very similar. For systems with metal ion:phytic acid molar ratios of 1:1–4:1 and 5:1–6:1 and pH values in the 3.4–5.9 and 3.4–5.0 ranges, respectively, Zn(II) ions were found to form complexes with phytic acid that were more stable than those of Cu(II) ions with phytic acid. The phytic acid molecule, however, was found to accommodate Cu(II) ions more readily than Zn(II) ions. For example, in systems containing equal amounts of Cu(II) and Zn(II) ions, 2 Zn(II) ions and 2, 3, 4, or 4.5 Cu(II) ions were found per phytic acid molecule depending upon metal ion:phytic acid molar ratios in the systems and pH. Total metal ion:phytic acid molar ratios and pH affected resultant metal ion solubilities and were factors influencing the effects of Zn(II) and Cu(II) ions on the binding of each other by phytic acid. Zn(II) and Cu(II) ions were observed to potentiate the binding of each other by phytic acid in some systems and compete with each other for phytate binding sites in others.     

The effects of monovalent cations Li+, Na+, K+, NH4+, Rb+ and Cs+ on the solid and solution structures of the nucleic acid components. Metal ion binding and sugar conformation.

The interactions of the monovalent ions Li+, Na+, K+, NH4+, Rb+ and Cs+ with adenosine-5'-monophosphoric acid (H2-AMP), guanosine-5'-monophosphoric acid (H2-GMP) and deoxyguanosine-5'-monophosphoric acid (H2-dGMP) were investigated in aqueous solution at physiological pH. The crystalline salts M2-nucleotide.nH2O, where M = Li+, Na+, K+ NH4+, Rb+ and Cs+, nucleotide = AMP, GMP and dGMP anions and n = 2-4 were isolated and characterized by Fourier Transform infrared (FTIR) and 1H-NMR spectroscopy. Spectroscopic evidence showed that these ions are in the form of M(H2O)n+ with no direct metal-nucleotide interaction, in aqueous solution. In the solid state, Li+ ions bind to the base N-7 site and the phosphate group (inner-sphere), while the NH4+ cations are in the vicinity of the N-7 position and the phosphate group, through hydrogen bonding systems. The Na-nucleotides and K-nucleotides are structurally similar. The Na+ ions bind to the phosphate group of the AMP through metal hydration shell (outer-sphere), whereas in the Na2-GMP, the hydrated metal ions bind to the base N-7 or the ribose hydroxyl groups (inner-sphere). The Na2-dGMP contains hydrated metal-carbonyl and metal-phosphate bindings (inner-sphere). The Rb+ and Cs+ ions are directly bonded to the phosphate groups and indirectly to the base moieties (via H2O). The ribose moiety shows C2'-endo/anti conformation for the free AMP acid and its alkali metal ion salts. In the free GMP acid, the ribose ring exhibits C3'-endo/anti conformer, while a C2'-endo/anti sugar pucker was found in the Na2-GMP and K2-GMP salts and a C3'-endo/anti conformation for the Li+, NH4+, Rb+ and Cs+ salts. The deoxyribose has C3'-endo/anti conformation in the free dGMP acid and O4'-endo/anti in the Na2-dGMP, K2-dGMP and a C3'-endo/anti for the Li+, NH4+, Rb+ and Cs+ salts. An equilibrium mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers was found for these metal-nucleotide salts in aqueous solution.     

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.     

An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically modified heparin, and heparin-derived oligosaccharides

The diprotonated form of histamine binds site-specifically to heparin, a highly sulfated 1-->4 linked repeating copolymer comprised predominantly of 2-O-sulfo-alpha-L-iduronic acid (the I ring) and 2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl (the A ring). The binding is mediated by electrostatic interactions. The structural features of histamine and heparin, which are required for the site-specific binding, have been identified from the results of (1)H NMR studies of the binding of histamine by six heparin-derived oligosaccharides and four chemically modified heparins and molecular modeling studies. The results indicate that the imidazolium ring of diprotonated histamine is critical for directing site-specific binding, while the ammonium group increases the binding affinity. The imidazolium ring binds within a cleft, with the A ring of an IAI triad at the top of the cleft, and the I rings forming the two sides. The H3 proton of the A ring is in the shielding cone of the imidazolium ring. The carboxylate group of the I-ring at the reducing end of the IAI triad and possibly the sulfamido group of the A-ring are essential for site-specific binding, whereas the 2-O-sulfate group of the I ring and the 6-O-sulfate group of the A ring are not. The results indicate that histamine binds to the IAI triad with the I rings in the (1)C(4) conformation. Also, the configuration of the carboxylate group is critical, as indicated by the absence of site-specific binding of histamine by the related IAG sequence, where G is alpha-D-glucuronic acid. The molecular modeling results indicate that the N1H and N3H protons of the imidazolium ring of site-specifically bound histamine are hydrogen bonded to the carboxylates of the I rings at the nonreducing and reducing ends of the IAI trisaccharide sequence.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Coordination chemistry of vitamin C. Part II. Interaction of L-ascorbic acid with Zn(II), Cd(II), Hg(II), and Mn(II) ions in the solid state and in aqueous solution

The reaction of L-ascorbic acid with the zinc group and manganese ions has been investigated in aqueous solution at pH 6-7. The solid salts of the type M (L-ascorbate)2.2H2O, where M = Zn(II), Cd(II) and Mn(II) were isolated and characterized by 13C NMR and Fourier Transform infrared (FT-IR) spectroscopy. Spectroscopic evidence showed that in aqueous solution, the bindings of the Zn(II) and Mn(II) ions are through the ascorbate anion O-3 and O(2)-H groups (chelation), while the Cd(II) ion binding is via the O-3 atom only. In the solid state, the binding of these metal ions would be through two acid anions via O-3, O-2 of the first and O-1, O-3 of the second anion as well as to two H2O molecules, resulting in a six-coordinated metal ion. The Hg(II) ion interaction leads to the oxidation of the ascorbic acid in aqueous solution.     

Conformational studies of A23187 with mono-, di- and trivalent metal ions by circular dichroism spectroscopy

CD studies carried out on A23187 indicate a solvent-dependent conformation for the free acid. Alkali metal ions were found to bind to the ionophore weakly. Divalent metal ions such as Mg2+, Ca2+, Sr2+, Ba2+ and Co2+ and trivalent lanthanide metal ions like La3+ were found to form predominantly 2:1 (ionophore-metal ion) complexes at low concentrations of metal ions, but both 2:1 and 1:1 complexes were formed with increasing salt concentration. Mg2+ and Co2+ exhibit similar CD behaviour that differs from that observed for the other divalent and lanthanide metal ions. The structure of 2:1 complexes involves two ligand molecules coordinated to the metal ion through the carboxylate oxygen, benzoxazole nitrogen and keto-pyrrole oxygen from each ligand molecule along with one or more solvent molecules. Values of the binding constant were determined for 2:1 complexes of the ionophore with divalent and lanthanide metal ions.     

Functional binding analysis of human fibrinogen as an iron- and heme-binding protein

Human fibrinogen is a metal ion-binding protein, but its mechanism of binding with iron and heme has not been elucidated in detail. In this study, human fibrinogen was immobilized on CNBr-activated Sepharose 4B beads. The fibrinogen beads bound hemin (iron–protoporphyrin IX: PPIX) as well as iron ion released from ferrous ammonium sulfate (FAS) more efficiently than Sepharose 4B beads alone. Hemin bound to fibrinogen still exhibited pseudo-peroxidase activity. The affinity of fibrinogen binding to hemin, Sn–PPIX, Zn–PPIX and metal-free PPIX followed the order Sn–PPIX < metal-free PPIX < hemin < Zn–PPIX; PPIX bound more non-specifically to control beads. FAS significantly enhanced the binding of hemin to fibrinogen beads. These results suggest that human fibrinogen directly recognizes iron ion, the PPIX ring and metal ions complexed with the PPIX ring, and that the binding of hemin is augmented by iron ions.     

Role of metal ions in catalysis by enolase: an ordered kinetic mechanism for a single substrate enzyme.

Spectroscopic and kinetic methods have been used to explore the roles of divalent metal ions in the enolase-catalyzed dehydration of 2-phosphoglycerate (2-PGA). Enolase requires 2 equiv of metal ion per active site for maximal activity. Previous crystallographic studies [Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) Biochemistry 35, 4349-4358] showed that both magnesium ions coordinated to the carboxylate group of the substrate/product-a scheme consistent with metal ion assistance in formation of the enolate intermediate. Electron paramagnetic resonance (EPR) data with 17O-labeled forms of phosphoenolpyruvate show that Mn(2+), bound at the lower affinity site, coordinates to one carboxylate oxygen and one phosphate oxygen of the substrate. These observations are fully consistent with the crystallographic data. Plots of activity versus log [metal ion] are bell-shaped, and the inhibitory phases of the profiles have been previously attributed to binding of metal ions at ancillary sites on the enzyme. However, the activation profiles and measurements of 2H kinetic isotope effects support an ordered kinetic mechanism wherein binding of 2-PGA precedes binding of the second metal ion, and release of the second metal ion occurs prior to departure of phosphoenolpyruvate. High concentrations of metal ion lead to inhibition in the ordered mechanism by interfering with product release. The 2H kinetic isotope effect is diminished in the inhibitory phases of the metal ion activation profiles in a manner that is consistent with the predominantly ordered mechanism. Zn(2+) gives lower maximal activity than Mg(2+), apparently due to slow release of Zn(2+) from the product complex. Addition of imidazole increases the maximal rate apparently by accelerating the release of Zn(2+) from the enzyme.     

Interaction of the polyelectrolyte heparin with copper(II) and calcium

The binding of copper(II) ions by heparin was investigated using equilibrium dialysi techniques, and the effects of this binding on solution properties determined. In neus tral Tris buffer solutions, heparin binds a maximum of twenty-three to twenty-four copper ions in two classes of sites, one containing three to four binding sites, the other containing twenty to twenty-one sites. Cooperative binding is associated with the larger class of sites. In more acidic citrate buffer solution, only one class of sites is observed, containing about four to five binding sites. Association constants are calculated for the classes and the possible chemical nature of the sites is discussed. The binding of calcium ions in neutral buffer is also examined, and these ions appear to be bound by a group of twenty to twenty-one binding sites, with a larger association constant than that for the copper ions. Definite effects on the solution properties of heparin, such as intrinsic viscosity, sedimentation coefficients, and partial specific volume, can be observed only in the cooperative binding of copper ions in neutral buffer. The interpretation of these solution properties in terms of molecular size and shape is analyzed, and it is concluded that the metal ion interactions cause no major change in the apparently random coil conformation of heparin in buffered solution, although some minor changes can be associated with the cooperative uptake of copper ions.     

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.     

The quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis exhibits a leaving group thio effect

Lactone-hydrolyzing enzymes derived from some Bacillus species are capable of disrupting quorum sensing in bacteria that use N-acyl-l-homoserine lactones (AHLs) as intercellular signaling molecules. Despite the promise of these quorum-quenching enzymes as therapeutic and anti-biofouling agents, the ring opening mechanism and the role of metal ions in catalysis have not been elucidated. Labeling studies using (18)O, (2)H, and the AHL lactonase from Bacillus thuringiensis implicate an addition-elimination pathway for ring opening in which a solvent-derived oxygen is incorporated into the product carboxylate, identifying the alcohol as the leaving group. (1)H NMR is used to show that metal binding is required to maintain proper folding. A thio effect is measured for hydrolysis of N-hexanoyl-l-homoserine lactone and the corresponding thiolactone by AHL lactonase disubstituted with alternative metal ions, including Mn(2+), Co(2+), Zn(2+), and Cd(2+). The magnitude of the thio effect on k(cat) values and the thiophilicity of the metal ion substitutions vary in parallel and are consistent with a kinetically significant interaction between the leaving group and the active site metal center during turnover. X-ray absorption spectroscopy confirms that dicobalt substitution does not result in large structural perturbations at the active site. Finally, substitution of the dinuclear metal site with Cd(2+) results in a greatly enhanced catalyst that can hydrolyze AHLs 1600-24000-fold faster than other reported quorum-quenching enzymes.     

Metal ion-induced conformational changes in Escherichia coli alkaline phosphatase.

Ultraviolet difference spectra are produced by the binding of divalent metal ions to metal-free alkaline phosphatase (EC 3.1.3.1). The interaction of the apoprotein with Zn2+, Mn2+, Co2+ and Cd2+, which induce the tight binding of one phosphate ion per dimer, give distinctly different ultraviolet spectra changes from Ni2+ and Hg2+ which do not induce phosphate binding. Spectrophotometric titrations at alkaline pH of various metallo-enzymes reveal a smaller number of ionizable tyrosines and a greater stability towards alkaline denaturation in the Zn2+- and Mn2+-enzymes than in the Ni2+-, Hg2+- and apoenzymes. The Zn2+- and Mn2+-enzymes have CD spectra in the region of the aromatic transitions that are different from the CD spectra of the Ni2+-, Hg2+- and apoenzymes. Modifications of arginines with 2,3-butanedione show that a smaller number of arginine residues are modified in the Zn2+-enzyme than in the Hg2+-enzyme. The presented data indicate that alkaline phosphatase from Escherichia coli must have a well-defined conformation in order to bind phosphate. Some metal ions (i.e. Zn2+, Co2+, Mn2+ and Cd2+), when interacting with the apoenzyme, alter the conformation of the protein molecule in such a way that it is able to interact with substrate molecules, while other metal ions (i.e. Ni2+ and Hg2+) are incapable of inducing the appropriate conformational change of the apoenzyme. These findings suggest an important structural function of the first two tightly bound metal ions in enzyme.     

The activation of concanavalin A by lanthanide ions.

Divalent cadmium and lead and the trivalent lanthanides bind in the trasition metal site (S1) of concamavanlin A and induce saccharide binding to the protein in the presence of calcium. Partial activation of the protein in the presence of lanthanides alone indicates these ions bind into both transition metal (S1) and calcium sites (S2). The activity of a lanthanide-protein derivative may be increased by the addition of either calcium or a transition metal ion. The saccharide binding activity decreases in the order Zn2+ is greater than Ni2+ is greater than Co2+ is greater than Mn2+ is greater than Cd2+ reflecting the order of binding constants for these ions in the transition metal site. Like the lanthanides, divalent cadmium substitutes for both the transition metal ion and calcium ion to partially activate the protein. Divalent lead substitutes only for the transition metal ion and partially activates the protein upon addingcalcium. The data are consistent with a model in which saccharide binding activity is independent of the metal size in S1 but critically dependent upon metal size in S2.     

Substrate specificity,metal binding properties,and spectroscopic characterization of the DapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae

The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to approximately 20% of the Zn(II)-loaded enzyme. The order of the observed k(cat) values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) approximately equal Cu(II) approximately equal Mg(II). DapE was shown to only hydrolyze L,L-N-succinyl-diaminopimelic acid (L,L-SDAP) and was inactive toward D,L-, L,D-, and D,D-SDAP. DapE was also inactive toward several acetylated amino acids as well as D,L-succinyl aminopimelate, which differs from the natural substrate, L,L-SDAP, by the absence of the amine group on the amino acid side chain. These data imply that the carboxylate of the succinyl moiety and the amine form important interactions with the active site of DapE. The affinity of DapE for one versus two Zn(II) ions differs by nearly 2.2 x 10(3) times (K(d1) = 0.14 microM vs K(d2) = 300 microM). In addition, an Arrhenius plot was constructed from k(cat) values measured between 16 and 35 degrees C and was linear over this temperature range. The activation energy for [ZnZn(DapE)] was found to be 31 kJ/mol with the remaining thermodynamic parameters calculated at 25 degrees C being DeltaG(++) = 64 kJ/mol, DeltaH(++) = 28.5 kJ/mol, and DeltaS(++) = -119 J mol(-1) K(-1). Electronic absorption and EPR spectra of [Co_(DapE)] and [CoCo(DapE)] indicate that the first Co(II) binding site is five-coordinate, while the second site is octahedral. In addition, any spin-spin interaction between the two Co(II) ions in [CoCo(DapE)] is very weak. The kinetic and spectroscopic data presented herein suggest that the DapE from H. influenzae has similar divalent metal binding properties to the aminopeptidase from Aeromonas proteolytica (AAP), and the observed divalent metal ion binding properties are discussed with respect to their catalytic roles in SDAP hydrolysis.