Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.     

113Cd NMR. Arsenate binding to Cd(II) alkaline phosphatase

Alkaline phosphatase from Escherichia coli contains three metal binding sites (A, B, and C) located at sites forming a triangle with sides of 4, 5, and 7 A (Wyckoff, H.W., Handschumacher, M., Murthy, K., and Sowadski, J.M. (1983) Adv. Enzymol. 55, 453). When all three sites are occupied by Cd(II) the enzyme has a very low turnover; at least 10(3) slower than the native Zn(II) enzyme. The slow turnover number has made the Cd(II) enzyme useful in NMR studies of the mechanism of alkaline phosphatase. The binding of arsenate to two forms of Cd(II) alkaline phosphatase (Cd(II)2alkaline phosphatase and Cd(II)6alkaline phosphatase) has been studied by 113Cd NMR. Cd(II)2alkaline phosphatase, pH 6.3, binds arsenate at only one monomer of the dimeric enzyme and causes migration of Cd(II) from the A site of one monomer to the B site of the arsenylated monomer. This same migration has previously been observed to accompany metal ion-dependent phosphate binding, but is much more rapid in the case of arsenate. The acceleration of migration induced by arsenate supports the conclusion based on the phosphate data that the substrate anion binds to the A site metal ion of one monomer prior to migration and that only the metal ion at A site is required for phosphorylation (arsenylation) of serine 102. The 113Cd chemical shifts of A and B site metal ions are very sensitive to the form of the bound arsenate, i.e. covalent (E-As) or noncovalent (E X As) complex. Like the analogous phosphate derivatives, the change of chemical shift of A site (to which phosphate is coordinated in the E X P complex) is much greater than that of the B site metal ion, when the arsenate shifts between the two intermediates, suggesting that arsenate is also coordinated to A site in the E X As intermediate. The chemical shifts of A and B site 113Cd(II) ions are considerably different in the arsenate and phosphate derivatives, while the C site 113Cd(II) ions have nearly identical chemical shifts. Thus the substrate appears to interact closely with both A and B sites, while C site appears relatively unimportant in phosphomonoester hydrolysis. The analogous behavior of arsenate and phosphate at the active center as evaluated by 113Cd NMR supports the validity of using the heavier arsenate derivative in x-ray diffraction studies.     

Alkaline phosphatase. 31P NMR probes of the mechanism

31P NMR signals from substrates and products of alkaline phosphatase have been adapted to measure the rates and product ratios for the hydrolysis and phosphotransferase reactions from pH 6 to 10. Below pH 8, glycerol is a poorer acceptor than H2O (glycerol phosphates:Pi = 0.5). Tris is a more effective acceptor below pH 8, showing a maximum acceptor efficiency at pH 8 (Tris phosphate:Pi = 2). Phosphotransferase efficiencies are in the order expected for the pKaS of the alcohol groups, Tris less than glycerol Cl, C3 less than glycerol C2. Tris and glycerol induce chemical shifts in 113Cd(II) present at the A site but not the B or C sites of the metal triad present at each active center of Cd(II)6 alkaline phosphatase, suggesting that the alcoxides of the acceptors coordinate the A site metal and become the nucleophiles attacking the phosphoseryl residue (E-P) in the second step of the mechanism. The interaction is through the oxygen of Tris. The transferase activity of the amino alcohol shows a bell-shaped pH dependency. Aliphatic alcohol acceptors show small increases in acceptor activity between pH 6 and 8, with 5-fold increases from pH 8 to 10 (at pH 10, glycerol phosphates:Pi = 2.5). 31P NMR inversion transfer has been used to measure the koff for Pi dissociation from the noncovalent enzyme complex (E . P). For the Zn(II)4 alkaline phosphatase koff is essentially pH independent at approximately 35 s-1. For Cd(II) or Mg(II) at the B site in place of Zn(II), koff less than or equal to 1 s-1 X Cl-ion, which appears to coordinate the A site metal ion, enhances koff, suggesting that both Cl- and HPO2-4 can coordinate the A site metal ion in a 5-coordinate intermediate. pH control of the alkaline phosphatase mechanism appears to reside in the stability of E-P and not the dissociation of E . P, compatible with the hypothesis that the activity-linked pKa is that of a H2O molecule coordinated to the A site metal, which in the hydroxide form becomes the nucleophile attacking the phosphoseryl group (E-P).     

113Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases

113Cd NMR spectra of 113Cd(II)-substituted Escherichia coli alkaline phosphatase have been recorded over a range of pH values, levels of metal site occupancy, and states of phosphorylation. Under all conditions resonances attributable to cadmium specifically bound at one or more of the three pairs of metal-binding sites (A, B, and C sites) are detected. By following changes in both the 113Cd and 31P NMR spectra of 113Cd(II)2 alkaline phosphatase during and after phosphorylation, it has been possible to assign the cadmium resonance that occurs between 140 and 170 ppm to Cd(II) bound to the A or catalytic site of the enzyme and the resonance occurring between 51 and 76 ppm to Cd(II) bound to B site, which from x-ray data is located 3.9 A from the A site. The kinetics of phosphorylation show that cadmium migration from the A site of one subunit to the B site of the second subunit follows and is a consequence of phosphate binding, thus precluding the migration as a sufficient explanation for half-of-the-sites reactivity. Rather, there is evidence for subunit-subunit interaction rendering the phosphate binding sites inequivalent. Although one metal ion, at A site, is sufficient for phosphate binding and phosphorylation, the presence of a second metal ion at B site greatly enhances the rate of phosphorylation. In the absence of phosphate, occupation of the lower affinity B and C sites produces exchange broadening of the cadmium resonances. Phosphorylation abolishes this exchange modulation. Magnesium at high concentration broadens the resonances to the point of undetectability. The chemical shift of 113Cd(II) in both A and B sites (but not C site) is different depending on the state of the bound phosphate (whether covalently or noncovalently bound) and gives separate resonances for each form. Care must be taken in attributing the initial distribution of cadmium or phosphate in the reconstituted enzyme to that of the equilibrium species in samples reconstituted from apoenzyme. Both 113Cd NMR and 31P NMR show that some conformational changes consequent to metal ion or phosphate binding require several days before the final equilibrium species is formed.     

31P NMR of alkaline phosphatase. Saturation transfer and metal-phosphorus coupling.

The rate constants which characterize the formation and breakdown of the noncovalent (E.P) and covalent (E-P) enzyme-phosphate intermediates on the alkaline phosphatase reaction pathway are known to be sensitive to the nature of the metal ion bound to the enzyme. 31P NMR saturation transfer has been demonstrated to provide a simple and sensitive method for measuring the metal ion dependence of these rates under equilibrium conditions. When the native Zn2+ was replaced by Cd2+, the 31P NMR spectrum at high pH revealed a new resonance at 12.6 ppm which has been assigned to the noncovalent enzyme.phosphate complex. Reconstituting the enzyme with enriched 113Cd2+ caused this unusually downfield-shifted resonance to appear as a doublet due to 113Cd-31P spin coupling (2J31P-O-113Cd = 30 Hz). This result provides the first unequivocal evidence for direct metal-phosphate interaction in alkaline phosphatase.     

31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH.

31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase. Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented. Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex. The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.     

Differential scanning calorimetry of Cd(II) alkaline phosphatases

Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme). Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion. Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively). Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition. These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy. These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme. Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C). This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C). The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM. These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme. This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples. These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding.     

1H, 113Cd, and 31P NMR of osteocalcin (bovine gamma-carboxyglutamic acid containing protein)

The 1H (500-MHz), 113Cd (44-MHz), and 31P (81-MHz) NMR spectra of the bovine gamma-carboxyglutamate- (Gla-) containing protein osteocalcin and its Ca(II) and Cd(II) complexes in solution have been obtained. The 1H NMR spectrum of the native protein shows narrow resonances and a highly resolved multiplet structure suggesting rotational freedom of the side chains. In comparison to the simulated 1H NMR spectrum of a random polypeptide chain of the same amino acid composition, there is moderate chemical shift dispersion, indicating some conformational restraints to be present. Ca(II) binding broadens all 1H resonances, so severely at four Ca(II) ions per molecule that few structural conclusions can be made. Cd(II) substituted for Ca(II) has the same effect, and 113Cd NMR shows the Cd(II) to be in intermediate chemical exchange on the chemical shift time scale. Estimates of the chemical exchange rates required for 1H and 113Cd line broadening suggest a range of Kd values for the metal ion complexes from 10(-6) M to as high as 10(-3) M depending on the number of metal ions bound. Alternatively, 1H line broadening could be explained by relatively slow conformational fluxes in the protein induced by labile metal ion binding to one or more sites. Cd(II) when used to form a cadmium-phosphate mineral analogous to hydroxylapatite results in a crystal lattice that removes osteocalcin from solution just as effectively as hydroxylapatite. 113Cd(II) exchange at the binding sites of osteocalcin in solution is slowed dramatically by the addition of HPO4(2-). 31P NMR shows the interaction of phosphate with the protein to require the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR of phosphate and phosphonate complexes of metalloalkaline phosphatases.

31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods. One equivalent of P1i, bound to Zn(II) alkaline phosphatase, pH 8, gives rise to a single 31P resonance 2 ppm downfield from that for Pi, and assignable to the noncovalent complex, E-P. Inorganic phosphate in excess of 1 eq per enzyme dimer gives rise to a resonance at the position expected for free Pi. At pH 5.1, a second resonance appears 8.5 ppm downfield from that for free Pi, and is assignable to the covalent complex, E-P. The large downfield shift suggests that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees.     

113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.     

Metal binding to Saccharomyces cerevisiae ferrochelatase

Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway. It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX. The crystal structures of ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work. The enzyme is a homodimer, with clear asymmetry between the monomers with regard to the porphyrin binding cleft and the mode of metal binding. The Co(II) and Cd(II) complexes reveal the metal binding site which consists of the invariant amino acids H235, E314, and S275 and solvent molecules. The shortest distance to the metal reveals that amino acid H235 is the primary metal binding residue. A second site with bound Cd(II) was found close to the surface of the molecule, approximately 14 A from H235, with E97, H317, and E326 participating in metal coordination. It is suggested that this site corresponds to the magnesium binding site in Bacillus subtilis ferrochelatase. The latter site is also located at the surface of the molecule and thought to be involved in initial metal binding and regulation.     

The metal binding site of zoocin A

Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity. A major (113)Cd resonance at 113.6 ppm was observed which with time split into resonances at 113.6 and 107.2 ppm. A minor (113)Cd resonance at 87.3 ppm was observed which increased in intensity with time. These Cd chemical shifts are indicative of two N atoms and two O atoms ligating directly to the metal site.On the basis of conserved amino acid residues in a homologous protein of known structure, LytM, the ligands in zoocin are tentatively assigned to H45, D49, H133, and some combination of water or buffer ions as the fourth oxygen donor in zoocin A. Comparison of the combined intensities for (113)Cd-substituted zoocin with a known quantity of another Cd-substituted protein gave Cd binding as approximately stoichiometric (1.2 +/- 0.2) with protein. Additional metal-removal and reconstitution experiments on the recombinant catalytic domain of zoocin implicate Zn(2+) as the metal cofactor. Therefore, the evidence supports zoocin as a single Zn(2+) ion binding metalloenzyme.     

13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin

13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.     

113Cd NMR studies on metal-thiolate cluster formation in rabbit Cd(II)-metallothionein: evidence for a pH dependence

The formation of two metal-thiolate clusters in rabbit liver metallothionein 2 (MT) has been examined by 113Cd NMR spectroscopy at pH 7.2 and 8.6. The chemical shifts of the 113Cd resonances developing in the course of apoMT titration with 113Cd(II) ions have been compared with those of fully metal occupied 113Cd7-MT. At pH 7.2 and at low metal occupancy (less than 4), a cooperative formation of the four-metal cluster (cluster A) occurs. Further addition of 113Cd(II) ions generates all the resonances of the three-metal cluster (cluster B) in succession, suggesting cooperative metal binding to this cluster also. In contrast, similar studies at pH 8.6, at low metal occupancy (less than 4), reveal a broad NMR signal centered at 688 ppm. This observation indicates that an entirely different protein structure exists. When exactly 4 equiv of 113Cd(II) are bound to apoMT, the 113Cd NMR spectrum changes to the characteristic spectrum of cluster A. Further addition of 113Cd(II) ions again leads to the cooperative formation of cluster B. These results stress the determining role of the cluster A domain on the overall protein fold. The observed pH dependence of the cluster formation in MT can be rationalized by the different degree of deprotonation of the cysteine residues (pKa approximately 8.9), i.e., by the difference in the Gibbs free energy required to bind Cd(II) ions to the thiolate ligands at both pH values.     

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.     

19-F NMR studies of the binding of a fluorine-labeled phosphonate ion to E. coli alkaline phosphatase.

The interaction of a fluorinated phosphonate with Zn-2+-and Mn-2+-alkaline phosphatase as studied by 19-F NMR revealed a stoichiometry of 1:1 for the binding of the phosphonate anion to the enzyme. In the presence of two metal ions, one fluorinated phosphonate ion was found to interact strongly with the enzyme, while a different interaction was observed when the number of metal ions per enzyme exceeded two. Phosphate replaced enzyme bound phosphonate, as is shown by the 19-F NMR spectra. No direct interaction between the fluorinated phosphonate and the metal ion responsible for enzyme activity was indicated by the 19-F NMR data. This observation supports the idea of a considerable distance between metal ion and substrate binding site in Escherichia coli alkaline phosphatase.     

Role of metal ions in the T- to R-allosteric transition in the insulin hexamer.

The role of metal ions in the T- to R-allosteric transition is ascertained from the investigation of the T- to R-allosteric transition of transition metal ions substituted-insulin hexamers, as well as from the kinetics of their dissociation. These studies establish that ligand field stabilization energy (LFSE), coordination geometry preference, and the Lewis acidity of the metal ion in the zinc sites modulate the T- to R-state transition. (1)H NMR, (113)Cd NMR, and UV-vis measurements demonstrate that, under suitable conditions, Fe2+/3+, Ni2+, and Cd2+ bind insulin to form stable hexamers, which are allosteric species. (1)H NMR R-state signatures are elicited by addition of phenol alone in the case of Ni(II)- and Cd(II)-substituted insulin hexamers. The Fe(II)-substituted insulin hexamer is converted to the ferric analogue upon addition of phenol. For the Fe(III)-substituted insulin hexamer, appearance of (1)H NMR R-state signatures requires, additionally to phenol, ligands containing a nitrogen that can donate a lone pair of electrons. This is consistent with stabilization of the R-state by heterotropic interactions between the phenol-binding pocket and ligand binding to Fe(III) in the zinc site. UV-vis measurements indicate that the (1)H NMR detected changes in the conformation of the Fe(III)-insulin hexamer are accompanied by a change in the electronic structure of the iron site. Kinetic measurements of the dissociation of the hexamers provide evidence for the modulation of the stability of the hexamer by ligand field stabilization effects. These kinetic studies also demonstrate that the T- to R-state transition in the insulin hexamer is governed by coordination geometry preference of the metal ion in the zinc site and the compatibility between Lewis acidity of the metal ion in the zinc site and the Lewis basicity of the exogenous ligands. Evidence for the alteration of the calcium site has been obtained from (113)Cd NMR measurements. This finding adds to the number of known conformational changes that occur during the T- to R-transition and is an important consideration in the formulation of allosteric mechanisms of the insulin hexamer.     

The cobalt(II)-alkaline phosphatase system at alkaline pH

The uptake of cobalt(II) ions by apoalkaline phosphatase at pH 8 (the pH optimum for activity) has been investigated by the combined use of electronic and 1H NMR spectroscopies. The presence of fast-relaxing high spin cobalt(II) ions in the active site cavity of the protein induces sizable isotropic shifts of the 1H NMR signals of metal-coordinated protein residues, allowing us to propose a metal uptake pattern by the various metal binding sites both in the presence and in the absence of magnesium ions. In the absence of magnesium the active site is not organized in specific metal binding sites. The first equivalent of cobalt(II) ions per dimer binds in an essentially unspecific and possibly fluxional fashion, giving rise to a six-coordinated chromophore. The second and third equivalents induce the formation of increasing amounts of metal ions pairs, cooperatively arranged into the A and B sites of the same subunit with a five- and six-coordinated geometry, respectively. The fourth and fifth equivalents induce the formation of fully blocked A-B pairs in both subunits. Magnesium shows the property of organizing the metal binding sites, probably through coordination to the C sites. Electronic and 1H NMR titration with Co2+ ions show that the initial amount of fluxional cobalt is smaller than in the absence of magnesium and that A-B pairs are more readily formed. Titration of fully metalated Co4Mg2alkaline phosphatase samples with phosphate confirms binding of only one phosphate per dimer.     

Characterization of the zinc binding site of bacterial phosphotriesterase.

The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity. This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents. In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium. The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy. The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days. Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+. The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2. The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers.     

Metallophosphoryl and Apophosphoryl Alkaline Phosphatases.

The noncovalent phosphate (E-P) and covalent phosphory (E-P) complexes of Zn(II), Cd(II), and apoalkaline phosphatases of Escherichia coli have been studied by stopped flow kinetic methods and 32P-labeling techniques. With 2,4-dinitrophenylphosphate as substrate, preincubation of the Zn(II) enzyme with Pi at pH 8 slows the pre-steady state burst rate, but does not affect the burst magnitude of 1 mol of ROH per enzyme dimer. Preincubation of the enzyme with Pi at pH 5.5 reduces the burst magnitude by one-half, as well as reducing the burst rate. Reduction of the burst magnitude as a function of the pH of the preincubation with Pi follows the same function as that previously established for the formation of E-P. Hence, ROP phosphorylates the enzyme by displacing phosphate from E-P during a pre-steady state reaction, while E-P turns over at the steady state velocity.