Nuclear magnetic resonance studies on the role of intrinsic metals in Escherichia coli RNA polymerase. Effect of DNA template on the nucleotide-enzyme interaction

Nuclear magnetic resonance studies were performed to investigate the effect of DNA template on the interaction of initiating nucleotide ATP with Escherichia coli RNA polymerase (RPase) in which one of the two intrinsic Zn ions was substituted with a Co(II) (Co-Zn RPase) or Mn(II) (Mn-Zn RPase) ion. This intrinsic metal ion is located at the initiation site in the beta subunit of RPase. The paramagnetic effects of Co-Zn and Mn-Zn RPases on the relaxation rates of 1H- and 31P-nuclei of ATP were used to determine the distances from the intrinsic metal to various atoms of ATP bound at the initiation sites in the presence of DNA. The distances from the metal to H2, H8, H1', alpha-P, beta-P, and gamma-P atoms were estimated to be 6.7 +/- 0.9, 4.1 +/- 0.6, 6.0 +/- 1.2, 7.5 +/- 0.8, 9.4 +/- 1.0, and 9.8 +/- 1.0 A, respectively. These distances were compared with those measured in the absence of DNA (Chatterji, D., and Wu, F. Y.-H. (1982) Biochemistry 21, 4657). In both the presence and absence of DNA, the close proximity between the intrinsic metal and the H8 atom strongly indicates that the metal is coordinated directly to the base moiety of ATP. Such a coordination may provide a structural basis for the selection of a purine nucleotide during the initiation process. The presence of DNA causes the H2 atom to move away (greater than 2 A) from the intrinsic metal, whereas all three phosphorus atoms shift closer (greater than 3 A) toward the metal. The possible mechanistic implications of the conformational alteration of ATP at the initiation site induced by the DNA template is discussed.     

7Li, 31P, and 1H NMR studies of interactions between ATP, monovalent cations, and divalent cation sites on rabbit muscle pyruvate kinase

The interactions between ATP, monovalent cations, and divalent cations on rabbit muscle pyruvate kinase have been examined using 7Li, 31P, and 1H nuclear magnetic resonance. Water proton nuclear relaxation studies are consistent with the binding of Li+ to the K+ site on pyruvate kinase with an affinity of 120 mM in the absence of substrates and 16 mM in the presence of P-enolpyruvate. Titrations with pyruvate demonstrate that pyruvate binds to the enzyme with an affinity of 0.65 mM in the presence of Li+ and 0.4 mM in the presence of K+. 7Li+ nuclear relaxation rates in solutions of pyruvate kinase are increased upon titration with the metal-nucleotide analogue, Cr(H2O)4ATP. Mn2+ EPR spectra were used to determined the distribution of the enzyme between the so-called isotropic and anisotropic conformations of the enzyme (Ash, D. E., Kayne, F., and Reed, G.H. Arch. Biochem. Biophys. (1978) 190, 571-577). Li-Cr distances of 5.6 and 11.0 A were calculated for the anisotropic and isotropic forms, respectively, in the absence or presence of pyruvate. When the divalent cation site on the enzyme was saturated with Mg2+, these distances increased to 6.7 and 9.5 A, respectively, regardless of the presence or absence of pyruvate. 31P nuclear relaxation studies with the diamagnetic metal-nucleotide analogue, Co(NH3)4ATP, indicated that addition of Mn2+ ion to the divalent cation site on the enzyme increased the longitudinal relaxation rates of all three phosphorus nuclei of the analogue. The 31P data indicate that the presence of pyruvate at the active site effects a decrease in the Mn-P distances, bringing Mn2+ and Co(NH3)4ATP closer together at the active site. The data also permit an evaluation of the role of the metal coordinated to the beta-P and gamma-P of ATP at the active site.     

Intrinsic zinc ion is essential for proper conformation of active Escherichia coli RNA polymerase

The DNA-dependent RNA polymerase (RPase) from Escherichia coli contained 2 mol of Zn/mol of holoenzyme (alpha 2 beta beta' sigma). An in vitro protocol involving sequential denaturation of RPase in 8 M urea and low pH (2.2), in the presence of 10 mM ethylenediaminetetraacetic acid (EDTA), was developed to completely remove the two intrinsic Zn ions. Subsequent reconstitution of the denatured, Zn-free RPase in the absence and presence of 10(-5) approximately 10(-4) M ZnCl2 yielded respectively the inactive apoenzyme and active (50 +/- 10%) RPase containing one Zn ion (rec-Zn1-RPase). Active rec-Cd1-RPase was similarly obtained when CdCl2 instead of ZnCl2 was used in the reconstitution. The use of 65Zn as a tracer in the two-step reconstitution procedure showed that the metal was incorporated into renatured enzyme only in the last step of reconstitution. The subunit location of the incorporated metal was identified to be in the beta subunit by the use of Affi-Gel Blue column chromatography of rec-Cd1-RPase. The analysis of apo- and rec-Zn1-RPases by sucrose density gradient sedimentation showed that the inactive apo-RPase appeared to be consisted of randomly folded protein species with S20,w values ranging from 5 to 18 S, while rec-Zn1-RPase contained a major, active 13S RPase species and a minor, inactive 7.9S species that could be separated by DNA-cellulose column chromatography. Both 13S and 7.9S RPase contained 1 mol of Zn and the five subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P and 1H NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: relaxation measurements with Mn(II) and Co(II)

The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the delta protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31P relaxation rates in E.MnADP and E.MnATP yields activation energies (delta E) in the range 6-10 kcal/mol. Thus, the 31P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-2 kcal/mol; i.e., these rates depend upon 31P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 A, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1H spin-lattice relaxation rate of the delta protons of arginine in the E.MnADP.Arg complex was also measured at three frequencies (viz., 200, 300, and 470 MHz). These 1H experiments were performed in the presence of sufficient excess of arginine to be observable over the protein background but with MnADP exclusively in the enzyme-bound form so that the enhancement in the relaxation rates of the delta protons of arginine arises entirely from the enzyme-bound complex. Both the observed frequency dependence of these rates and the delta E less than or equal to 1.0 +/- 0.3 kcal/mol indicate that this rate depends on the 1H-Mn(II) distances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding site of Fe3+ at purine of ATP as studied by NMR

The binding site of Fe3+ in the purine base of adenosine 5'-triphosphate (ATP) was studied by nuclear magnetic resonance (NMR). The NMR relaxation rates (R1) of 1H and 31P in ATP solutions free of and containing ferric ions were measured in the pH range of 3-10. It was found that Fe3+ selectively enhanced the relaxation rate of protons. In the presence of Fe3+, the R1 of H2 was much bigger than that of H8 at a lower pH (3-4.5), while at a higher pH (5.5-7.5) the R1 of H8 was more enhanced than H2. At a pH of around 5, both H2 and H8, as well as all three phosphorous, showed a sudden jump in R1. When pH>8, Fe3+ failed to show appreciable enhancement of R1 to all protons and phosphorous. The quantitative data of relaxation rate enhancements suggest that the binding site of Fe3+ in ATP is strongly dependent on pH. At lower pH values, Fe3+ binds N1 but at higher pH it binds to N7. When pH is around 5, the whole purine base donates the aromatic pi-electrons to the ferric ion, forming a ferrocene-like complex, while when pH>8, ATP could not form complexes with Fe3+.     

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.     

Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

How do MgATP analogues differentially modify high-affinity and low-affinity ATP binding sites of Na+/K(+)-ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.     

Effects of divalent cations on encapsulation and release in the GroEL-assisted folding

Chaperonin GroEL assists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL-GroES cis-ternary complexes, but prevent the release of METF from GroEL.     

Isolation and characterization of an amiloride-resistant mutant of Methanothermobacter thermautotrophicus possessing a defective Na+/H+ antiport

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the Na+/H+ antiporter inhibitor amiloride was isolated. The Na+/H+ exchanger activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Methanogenesis rates in the mutant strain were higher than wild-type cells and resistant to the inhibitory effect of 2 mM amiloride. In contrast, methanogenesis in wild-type cells was completely inhibited by the same amiloride concentration. ATP synthesis driven by methanogenic electron transport or by an electrogenic potassium efflux in the presence of sodium ions was significantly enhanced in the mutant cells. ATP synthesis driven by potassium diffusion potential was profoundly inhibited in wild-type cells by the presence of uncoupler 3,3',4',5- tetrachlorosalicylanilide and sodium ions, whereas c. 50% inhibition was observed in the mutant cells under the same conditions.     

Activation and inactivation of horseradish peroxidase by cobalt ions

The effect of cobalt ions (Co2+) on horseradish peroxidase (HRP) was studied in vitro by enzymatic activity assay, electronic absorption spectra, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate(ANS)-binding fluorescence spectra. Co2+ at concentrations below 0.1 mM mildly increased the HRP activity, whereas higher concentrations of Co2+ significantly inactivated HRP in a time and concentration-dependent manner. Steady-state kinetic studies show that Co2+ was a noncompetitive inhibitor of o-dianisidine oxidation by HRP. The Ki value dropped as the incubation time increased. Furthermore, Co2+ was found to be an uncompetitive inhibitor of H2O2. These results suggested that Co2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectroscopic analysis showed that even for high Co2+ concentrations, the structure of HRP as a whole only changed slightly; however, there were significant conformational changes near or in the active site of HRP. Based on the above results, we suggest that Co2+ may bind with some amino acids near or in the active site of HRP and the conformational changes of HRP induced by such binding should be the main reason for activation and inactivation effect of Co2+. The potential binding sites of Co2+ were also proposed.     

Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules

The possible presence of ATP-driven H+ translocase activity in isolated rat parotid secretory granules has been examined by several approaches. First the transmembrane pH difference measured by either [14C] methylamine or [3H]acetate distribution is not substantially affected by ATP in the presence of membrane-permeating anions. Second, despite a low intrinsic H+ permeability of parotid granule membranes, only a small variably detectable inside-positive transmembrane potential is observed (by altered distribution of radioactive ions) when ATP is added in the absence of permeant anions. Third, ATP-induced lysis of parotid granules is minor and appears to be independent of ATP hydrolysis. Finally, ATP-hydrolase activity of the parotid granule fraction is not stimulated by an H+ ionophore, nor is it susceptible to inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at a concentration which decreases the measured ATPase of purified chromaffin granule membranes by more than 80%. These findings suggest that this exocrine secretory granule type, which is characterized by storage of a heterogeneous mixture of secretory proteins, exhibits H+ pump activity which is at most a small fraction of that observed in biogenic amine storage granules of neural and endocrine tissues.     

Regulation of substrate specificity of plant alpha-mannosidase by cobalt ion: in vitro hydrolysis of high-mannose type N-glycans by Co2+-activated Ginkgo alpha-mannosidase

In our previous study (Woo, K. K., et al., Biosci. Biotechnol. Biochem., 68, 2547-2556 (2004), we purified an alpha-mannosidase from Ginkgo biloba seeds; it was activated by cobalt ions and highly active towards high-mannose type free N-glycans occurring in plant cells. In the present study, we have found that the substrate specificity of Ginkgo alpha-mannosidase is significantly regulated by cobalt ions. When pyridylamino derivative of Man9GlcNAc2 (M9A) was incubated with Ginkgo alpha-mannosidase in the absence of cobalt ions, Man5GlcNAc2-PA (M5A) having no alpha1-2 mannosyl residue was obtained as a major product. On the other hand, when Man9GlcNAc2-PA was incubated with alpha-mannosidase in the presence of Co2+ (1 mM), Man3-1GlcNAc2-PA were obtained as major products releasing alpha1-3/6 mannosyl residues in addition to alpha1-2 mannosyl residues. The structures of the products (Man8-5GlcNAc2-PA) derived from M9A by enzyme digestion in the absence of cobalt ions were the same as those in the presence of cobalt ions. These results clearly suggest that the trimming pathway from M9A to M5A is not affected by the addition of cobalt ions, but that hydrolytic activity towards alpha1-3/6 mannosyl linkages is stimulated by Co2+. Structural analysis of the products also showed clearly that Ginkgo alpha-mannosidase can produce truncated high-mannose type N-glycans, found in developing or growing plant cells, suggesting that alpha-mannosidase might be involved in the degradation of high-mannose type free N-glycans.     

Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli.

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.     

NMR relaxation measurements detect four intermediate states of ATPase and transport cycle of sarcoplasmic reticulum Ca2+-ATPase

At least four of the intermediate states of Ca2+-ATPase (and presumably ion transport) can be trapped and characterized using water proton relaxation measurements. Gd3+ binds to two occluded Ca2+ transport sites on Ca2+-ATPase which have a low accessibility to solvent water. In the presence of the MgATP analogue Co(NH3)4AMPPCP, a new state for bound Gd3+ with one less water of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected by NMR, the first of which probably represents an intermediate state of ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and corresponds to the highly occluded E1-P state observed with CrATP (Vilsen and Andersen, Biochim. Biophys. Acta 898, 313 (1987).     

31P NMR studies of the structure of cation-nucleotide complexes bound to porcine muscle adenylate kinase

The paramagnetic effects on the spin-relaxation rates of 31P nuclei in complexes of porcine muscle adenylate kinase with ATP, GTP, GDP, and AMP were measured in the presence of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), to examine the structures of the enzyme-bound complexes. Experiments were performed exclusively on enzyme-bound complexes to limit contributions to observed relaxation rates to two exchanging complexes (with and without cation). Measurements were made at three frequencies, 81, 121.5, and 190.2 MHz, and as a function of temperature in the range 5-30 degrees C to determine the effect of exchange on the observed relaxation rates. Relaxation rates in the E.MnATP, E.MnGTP, and E.MnGDP complexes were shown to be exchange-limited and therefore without structural information. Relaxation rates for the complexes E.CoATP, E.CoGTP, and E.CoGDP were shown to depend on Co(II)-31P distances. Inability to precisely estimate spectral densities arising from electronic relaxation of Co(II) restricts calculations of Co(II)-31P distances in these complexes to upper and lower limits. At the center of these limits, the Co(II)-31P distances of beta-P and gamma-P in E.CoATP and E.CoGTP, and of beta-P (E.CoGDP), are in the range 3.1-3.5 A appropriate for the first coordination sphere. For all these complexes, the corresponding distance for alpha-P is appreciably larger in the range 3.9-4.5 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and characterization of various Escherichia coli RNA polymerases containing one or two intrinsic metal ions

The Escherichia coli DNA-dependent RNA polymerase (RPase) holoenzyme (alpha 2 beta beta' sigma) possesses 2 mol equiv of Zn: beta and beta' subunits each contain one Zn ion. An in vitro metal-substitution method developed earlier (method I) was used to remove the two intrinsic Zn ions and then to reconstitute other metal ions into the beta subunit of RPase. One Cd or Hg ion was successfully reconstituted into half-active enzymes (rec-Cd1- or rec-Hg1-RPase), while Mn or Ni ion was not incorporated. A new, simplified in vitro metal-substitution method (method II), which omitted the low-pH treatment and subsequent urea dialysis in method I, was devised in this study. Consequently, Zn or Cd could be incorporated into both the beta and beta' subunits, resulting in rec-Zn2- or rec-Cd2-RPase, respectively. However, only one Hg was incorporated, probably due to steric hindrance by the large size of the Hg ion, while Mn, Ni, or Cr was not bound by the reconstituted enzyme, which instead incorporated only one Zn. Analysis of the metal content of various reconstituted RPases indicated that without low-pH treatment Zn bound to both the beta and beta' subunits when Zn concentrations were higher than 2 X 10(-6)M, but it bound only to the beta' subunit at lower concentrations. Moreover, low-pH treatment destroys the metal binding site in the beta' subunit. The metal sites on the beta and beta' subunits did not have significant affinity for the transition metals such as Mn, Ni, and Cr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective and effective bactericidal activity of the cobalt (II) cation against Helicobacter pylori

BACKGROUND: Although the anti-Helicobacter pylori activity of bismuth is well established, the therapeutic potential of other metal ions against the organism is not known. MATERIALS AND METHODS: We measured the minimum inhibitory concentrations of a series of metal ions, including several cobalt (II) compounds against four type strains and seven clinical isolates of H. pylori using three standard broth culture media and a defined medium. Other intestinal bacteria were also investigated for specificity of action. RESULTS: Cobalt chloride had marked activity against H. pylori (minimum inhibitory concentration range was 0.03-1.0 mg/l). The effect was specific because other transition metals had no effect and other intestinal bacteria were not affected by cobalt chloride. Activity was attributable to free cobalt ions as ligands inhibited activity in proportion to their affinity for the ions. Inhibition of cobalt activity was also observed in the presence of nickel, in a dose dependent fashion. However, cobalt activity was not directed towards the nickel-dependent urease enzyme because its effect was similar in wild-type and urease negative mutant strains of H. pylori. Finally, the viability of H. pylori was reduced at the same rate with 2 mg/l cobalt as with 1 mg/l amoxicillin. CONCLUSIONS: Cobalt competes for nickel in its acquisition by H. pylori, but mediates toxicity in a nonurease dependent fashion. As cobalt MIC is similar to some antibiotics and 10 to a hundred times lower than for bismuth, cobalt may represent an effective form of therapy for H. pylori infection.     

1H and 31P nuclear magnetic resonance and kinetic studies of the active site structure of chloroplast CF1 ATP synthase

The interaction of nucleotides and nucleotide analogues and their metal complexes with Mn2+ bound to both the latent and dithiothreitol-activated CF1 ATP synthase has been examined by means of steady-state kinetics, water proton relaxation rate (PRR) measurements, and 1H and 31P nuclear relaxation measurements. Titration of both the latent and activated Mn(2+)-CF1 complexes with ATP, ADP, Pi, Co(NH3)4ATP, Co(NH3)4ADP, and Co(NH3)4AMPPCP leads to increases in the water relaxation enhancement, consistent with enhanced metal binding and a high ternary complex enhancement. Steady-state kinetic studies are consistent with competitive inhibition of CF1 by Co(NH3)4AMPPCP with respect to CaATP. The data are consistent with a Ki for Co(NH3)4AMPPCP of 650 microM, in good agreement with a previous Ki of 724 microM for Cr(H2O)4ATP [Frasch, W., & Selman, B. (1982) Biochemistry 21, 3636-3643], and a best fit KD of 209 microM from the water PRR measurements. 1H and 31P nuclear relaxation measurements in solutions of CF1 and Co(NH3)4AMPPCP were used to determine the conformation of the bound substrate analogue and the arrangement with respect to this structure of high- and low-affinity sites for Mn2+. The bound nucleotide analogue adopts a bent conformation, with the low-affinity Mn2+ site situated between the adenine and triphosphate moieties and the high-affinity metal site located on the far side of the triphosphate chain. The low-affinity metal forms a distorted inner-sphere complex with the beta-P and gamma-P of the substrate. The distances from Mn2+ to the triphosphate chain are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules or residues from the protein.     

NMR studies identify four intermediate states of ATPase and the ion transport cycle of sarcoplasmic reticulum Ca2+-ATPase

Water proton nuclear relaxation measurements are used to detect and characterize four distinct intermediate states for Gd3+ bound to Ca2+ sites of sarcoplasmic reticulum Ca2+-ATPase in complexes with ATP analogues. In the absence of nucleotides, Gd3+ binds to two occluded Ca2+ transport sites on Ca2+-ATPase which have a low accessibility to solvent water. In the presence of the nonhydrolyzable ATP analogue, Co(NH3)4AMPPCP, a new state for bound Gd3+ (still occluded and with fewer waters of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected in the NMR studies. The first of these probably represents an intermediate state for bound Gd3+ during ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and is probably analogous to the highly occluded E1-P state observed with CrATP [(1987) Biochim. Biophys. Acta 898, 313-322].