The binding of copper ions to copper-free bovine superoxide dismutase. Copper distribution in protein samples recombined with less than stoicheiometric copper ion/protein ratios.

Samples of superoxide dismutase containing less than stoicheiometric amounts of Cu2+ were obtained by either partial re-addition of Cu2+ to the Cu2+-free protein or partial removal of Cu2+ by controlled CN-treatment. In these samples the distribution of the metal between the two identical sites on the two subunits was studied by quantitative gel electrophoresis and found to be statistical only in the process of copper removal by CN-. In the other case the distribution fits a model of co-operative interaction between the two sites, where the sites are equivalent for the binding of the first Cu2+ ion, but the occupation of the first site lowers the activation energy of the binding of the second Cu2+ ion. This indicates that binding of Cu2+ ion at its site on one subunit brings about conformational changes that facilitate Cu2+ binding on the other subunit. These results may relate to possible intersubunit interactions during the catalytic activity.     

The binding of copper ions to copper-free bovine superoxide dismutase. Kinetic aspects.

The kinetics of reconstitution of bovine superoxide dismutase from Cu2+ and the copper-free enzyme have been studied by activity, u.v.-absorption, electron-paramagnetic-resonance and pulsed-nuclear-magnetic-resonance measurements. The process appears to be first-order up to 80% completion in most conditions, and is pH-dependent, with an apparent pK of 6.5. U.v.-absorption and solvent proton relaxation rate measurements show that fast binding of Cu2+ occurs, and the initial ligands are likely to be, at least in part, those of the native active site. The recovery of the native activity and spectroscopic properties is a slow process with activation energies of 92 kJ/mol at pH 5.3 and 8.4kJ/mol at pH 8.1 and can be described as a rearrangement of the site around the bound metal. The rate of this process is lower in partially recombined protein samples, probably because of intersubunit interactions.     

Properties of cupric ions in benzylamine oxidase from pig plasma as studied by magnetic-resonance and kinetic methods.

Benzylamine oxidase from pig plasma has been studied by a variety of chemical and physical techniques. 1. Analytical ultracentrifugation, gel electrophoresis and isoelectric-focusing studies suggest that the enzyme is composed of two subunits with closely similar primary structures. 2. E.s.r. and n.m.r. measurements show that the enzyme contains two well-separated (greater than 0.6 nm) Cu2+ ions at chemically distinct sites. Each Cu2+ ion is coordinated by two water molecules, one 'axial' and the other 'equatorial'. Both water molecules undergo fast exchange (10(5)--10(8) s-1) with solvent and are deprotonated in the pH range 8--9, but only the equatorial water molecule is displaced by the inhibitors N3- and CN-. 3. Kinetic and e.s.r. measurements show that azide and cyanide compete against O2 binding and also make the two Cu2+ sites identical. It is concluded that Cu2+ must participate in the re-oxidation of reduced enzyme by molecular O2.     

Effect of the concentration ratio of bi- and monovalent ions on the functional activity of 30S ribosome subunits of Escherichia coli

Experiments on poly(U)-dependent binding of Phe-tRNAPhe to 30S subunits revealed the existence of a critical [Mg2+]/[NH4+] ratio in a medium (approximately 0.05-0.1) with respect to the binding capacity of subunits. If the ratio is greater than the critical one, 30S subunits undergo reversible inactivation even at the highest Mg2+ concentrations (up to 20 mM). The stronger is the deviation from the [Mg2+]/[NH4+] value = 0.05-0.1, the greater are both the rate and extent of such an inactivation. Two sites for tRNA in initially active 30S subunits have been shown to be inactivated in an interdependent way. On the other hand, a progressive decrease of [Mg2+]/[NH4+] ratio in a medium (from the value of 0.05 and lower) does not produce inactivation, but rather results in reduced affinity constants of Phe-tRNAPhe for active sites of 30S subunits.     

The dimerization of ferrihaems. I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization.

The dimerization of dueteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (Robs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, Robs greater than 2, whereas for solutions containing mainly dimeric species Robs less than 1. A computer programme has been applied to determine values of the dimerization constant, K, defined as: K = [dimer] [H+]/[monomer]2. Phosphate buffer anions and Tris . HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order K+ less than Na+ less than Li+ less than Sr2+ less than Mg2+ approximately or equal to Ca2+. Values of K were determined for several concentrations of Na+ and Sr2+. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation 'sandwiched' between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.     

The interaction of [Ru(NH3)5Cl]2+ and [Ru(NH3)6]3+ ions with DNA

The interaction of [Ru(NH3)5Cl]2+ and [Ru(NH3)6]3+ complex ions with calf thymus DNA has been studied at various r values (r = [Mn+]/[DNA-P]). Electronic spectra of metal-DNA solutions have been recorded and compared to the spectra of metal, as well as of DNA, solutions. Melting curves have been taken for the determination of DNA melting temperature (Tm) in the presence of the above complex ions. The results showed a biphasic melting of the DNA strands for relatively high r values. The Tm for the first phase increased with increasing r values, indicating metal ion interaction with the phosphate moieties of the DNA. The appearance of a second-phase melting, in connection with electronic spectra, pH values, and conductivity measurements of metal ion solutions, is indicative of the initial complexes' transformation to [Ru(NH3)5OH]2+, which binds preferentially to double-stranded rather than single-stranded DNA, thus leading to a second melting curve at a higher temperature than the first one.     

Studies on the reconstitution of bovine erythrocyte superoxide dismutase. V. Preparation and properties of derivatives in which both zinc and copper sites contain copper.

1. We have developed a procedure for preparing derivatives of bovine superoxide dismutase in which primarily the Cu binding sites are occupied by Cu2+ (2 Cu2+-) and in which both the Zn and Cu binding sites are occupied by Cu2+ (4 Cu2+-). 2. The 2 Cu2+ protein shows approximately one-half the superoxide dismutase activity of an equivalent amount of native protein. A two-fold enhancement of the activity of 2 Cu2+-dismutase was observed upon occupation of the Zn sites either with Zn2+ or Cu2+. 3. The electron paramagnetic resonance spectrum of 4 Cu2+ protein was recorded over the temperature range 5-100 degrees K and the results suggest an antiferro-magnetic interaction between Cu2+ in the Zn site and Cu2+ in the Cu site having a coupling constant of approx. 52 cm-1. 4. The binuclear Cu2+ complex was found to accept only one electron from ferrocyanide. 5. One-half the total Cu+ of dithionite reduced 4 Cu+ protein was found to react rapidly with bathocupreine sulfonate whereas the other half reacted slowly. Reduced native protein did not react with bathocupreine sulfonate below 70 degrees C.     

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.     

Effect of metal ions on the secondary structure and activity of calf intestine phosphatase

Cobalt is an essential microelements in many biological processes involving enzymatic activity. We found that Zn2+ and Mg2+, which are in the active site of native calf intestine alkaline phosphatase (CIP), can be replaced by Co2+ directly in solution. The effect of Co2+ concentration on the substitution reaction was examined at ratios of [Co2+]/[CIP] from 0:1 to 8:1. The quantity of Zn2+ in CIP decreased progressively as the ratio was increased, but the amount of Mg2+ changed in irrregular fashion. A series of active site models of the reaction mechanism of CIP are proposed. Low pH was found to promote the replacement of Mg2+ by Co2+. To understand how the substitution affects the enzyme, we also solved the secondary structure of CIP after reaction with Co2+ in different conditions.     

Tryptophan 2,3-dioxygenase: a review of the roles of the heme and copper cofactors in catalysis.

L-Tryptophan, 2,3-dioxygenase (EC 1.13.11.11) has been purified to homogenity from L-tryptophan induced Pseudomonas acidovorans (ATCC 11299b) and from L-tryptophan and cortisone induced rat liver. The enzyme from both sources is composed of four subunits and contains two g-atoms copper and two moles heme per mole tetramer. The proteins from the two sources are not identical. Three oxidation states of tryptophan oxygenase have been isolated: (1) fully oxidized, [Cu(II)]2[Ferriheme]2; (2) half reduced, [Cu(i)]2[ferriheme]2; and (3) fully reduced, [Cu(I)]2[ferroheme]2. Catalytic activity is dependent solely on the presence of Cu(I) in the enzyme, the heme may be either ferro or ferri. The presence of Cu(II) in the enzyme results in a requirement for an exogenous reductant, such as ascorbate, in order to elicit enzymic activity. Ligands, such as cyanide and carbon monoxide, can inhibit catalysis by binding to either or to both the copper and heme moieties. Metal complexing agents, such as bathocuproinesulfonate and bathophenanthrolinesulfonate, can inhibit catalysis by binding to Cu(I) resent only in catalytically active enzyme molecules. During catalysis by the fully reduced form of the enzyme, molecular oxygen binds to the heme moieties, while during catalysis by the half reduced form of the enzyme it does not, presumably binding instead to the Cu(I) moieties. Enzymes that catalyze similar reactions have been purified from other sources. Indoleamine 2,3-dioxygenase appears to be a heme protein, but its copper content is unknown. Pyrrolooxygenases appear to be completely different enzymes, although they have not yet been purified to homegeneity.     

Reactions of bovine serum amine oxidase with NN-diethyldithiocarbamate. Selective removal of one copper ion.

NN-Diethyldithiocarbamate (DDC) was able to bind, at 1.0 mM concentration, only about 50% the Cu(II) ions of bovine plasma amine oxidase. Under reducing conditions, this Cu(II) was removed with inactivation of the enzyme. Up to 90% activity could be recovered by treatment with excess Cu(II). The organic cofactor, sensitive to carbonyl reagents, was reduced in the half-Cu-depleted protein and no longer bound phenylhydrazine. The fully reacted protein, in the presence of 10 mM-DDC, lost 50% Cu(II) upon storage at -20 degrees C, but in this case the residual Cu(II) was in the DDC-bound form and the cofactor was in the oxidized state, as it could still bind phenylhydrazine. In the presence of DDC, the rate of reaction with phenylhydrazine was always low, even at 50% DDC saturation, and all derivatives showed identical modifications of the optical and e.p.r. spectra with respect to the phenylhydrazone of the native protein. It is concluded that the two Cu(II) ions are not equivalent, that removal of a single Cu(II) is sufficient to inhibit the re-oxidation of the organic cofactor, and that both Cu(II) ions are in some way involved in the reaction with phenylhydrazine. After reaction with DDC, the optical and e.p.r. spectra of 63Cu(II)-amine oxidase and of 63Cu(II)-carbonic anhydrase [Morpurgo, Desideri, Rigo, Viglino & Rotilio (1983) Biochim. Biophys. Acta 746, 168-175] are very similar and show distorted equatorial co-ordination to Cu(II) of two sulphur atoms and two magnetically equivalent nitrogen atoms.     

Effect of diphosphonic acids on alkaline phosphatase activity

Kinetics was studied for the alkaline phosphatase activity inhibition by diphosphonic acids. When the ratio of Mg2+ and substrate (S) concentrations [( Mg2+]/[S]) is equal to 10, the process constants for methylene diphosphonic, amino methylene diphosphonic and hydroxyethylidene diphosphonic acids are 0.14, 0.12 and 0.35 mM, respectively. The inhibition is of competitive character. An increase in the Mg2+ concentration to the [Mg2+]/[S] = 40 ratio lowers the inhibition degree for all three diphosphonates; it follows a mixed mechanism. Thus, the inhibition of the alkaline phosphatase activity by diphosphonic acids is due to both competition of the inhibitor for the enzyme active centre and a decrease in the Mg2+ concentration, the phosphatase activator, because of Mg2+ complexing with diphosphonates.     

Effects of calcium ion on ternary complexes formed between 4-(2-pyridylazo)resorcinol and the two-zinc insulin hexamer

As a means for probing the microenvironment of zinc in the insulin hexamer and to investigate the effects of calcium ion on the assembly and the structure of the two-zinc insulin hexamer, the thermodynamics and kinetics of the reaction between the chromophoric divalent metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) and zinc-insulin have been investigated over a wide range of conditions. For [PAR]0 much greater than [Zn2+]0 and [Zn2+]/[In] less than or equal to 0.33, the reaction leads to the sequestering and ultimate removal of all of the insulin-bound Zn2+; for [Zn2+]0 much greater than [PAR]0, two stable ternary complexes are formed where Zn2+ has ligands derived from PAR as well as from hexameric insulin. For [Zn2+]/[In] ratios below 0.33, the equilibrium distribution between the two ternary complexes is dependent on the [Zn2+]/[In] ratio. One of the complexes is assigned to the monoanion of PAR coordinated to Zn2+ that resides in a His-B10 site. The other complex is proposed to involve the coordination of (PAR)Zn to the site formed by the alpha-NH2 group of Phe-B1 and the gamma-carboxylate ion of Glu-A17 across the dimer-dimer interface on the surface of the hexamer. With either PAR or zinc-insulin in large excess, the kinetics of the PAR optical density changes are remarkably similar and biphasic. The faster step is first order in PAR and first order in insulin-bound Zn2+ (k congruent to 3 X 10(3) M-1 s-1) and involves the formation of an intermediate in which PAR is coordinated to insulin-bound zinc at the His-B10 site.(ABSTRACT TRUNCATED AT 250 WORDS)     

铜金属螯合载体固定糖化酶

[目的]制备出含Cu2+的琼脂糖-IDA螯合载体及对其固定糖化酶工艺条件进行优化.[方法]利用金属螯合配体(IDA-Cu2+)与蛋白质表面供电子氨基酸相互作用的原理制备载体,采用紫外分光光度法测定不同影响因素下固定化糖化酶的酶活.[结果]Cu2+的加入量和固定化过程的酸度比给酶量对固定化糖化酶的活性影响还要大,在给酶量80 mg/g载体、1.0× 10-2 mol Cu2+/g载体、pH 4.6和固定化4h的固定化条件下,固定化酶活为252.1 U/g,重复使用5次后酶活为首次固定化酶活的65.1％.[结论]该Cu2+-IDA-金属螯合琼脂糖可用于淀粉水解糖化酶的优良固定化载体材料.     

Characterization and structure of a Zn2+ and [2Fe-2S]-containing copper chaperone from Archaeoglobus fulgidus

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 A resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.     

Studies on the active site of pig plasma amine oxidase.

Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity.     

Pyruvate dehydrogenase activity in hamster small intestine during development.

Total pyruvate dehydrogenase activities in hamster intestine increase from 40 nmol/min (munits) per g of intestine in the foetal animals to 460 munits/g in the adult, whereas the fraction of the enzyme in the active form increases from 34 to 42% of the total activity over the same period. However, a complete conversion of the enzyme into the active form is observed in the neonatal animal immediately after birth. Results from experiments in vitro suggested that the activation of pyruvate dehydrogenase is controlled, in part, by the [NAD+]/[NADH] ratio. This proposal was tested in vivo by examining the proportion of the enzyme in the active form during conditions when the [NAD+]/[NADH] ratio was markedly altered, and the data show a direct relationship between the mitochondrial redox state and activity of the active form.     

Roles of the two copper ions in bovine serum amine oxidase

With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically identical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half-depBSAO were estimated to be 5 X 10(6), 5 X 10(4), and 1 X 10(5) M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.