Structural studies of metal ions in family II pyrophosphatases: the requirement for a Janus ion

Family II inorganic pyrophosphatases (PPases) constitute a new evolutionary group of PPases, with a different fold and mechanism than the common family I enzyme; they are related to the "DHH" family of phosphoesterases. Biochemical studies have shown that Mn(2+) and Co(2+) preferentially activate family II PPases; Mg(2+) partially activates; and Zn(2+) can either activate or inhibit (Zyryanov et al., Biochemistry, 43, 14395-14402, accompanying paper in this issue). The three solved family II PPase structures did not explain the differences between the PPase families nor the metal ion differences described above. We therefore solved three new family II PPase structures: Bacillus subtilis PPase (Bs-PPase) dimer core bound to Mn(2+) at 1.3 A resolution, and, at 2.05 A resolution, metal-free Bs-PPase and Streptococcus gordonii (Sg-PPase) containing sulfate and Zn(2+). Comparison of the new and old structures of various family II PPases demonstrates why the family II enzyme prefers Mn(2+) or Co(2+), as an activator rather than Mg(2+). Both M1 and M2 undergo significant changes upon substrate binding, changing from five-coordinate to octahedral geometry. Mn(2+) and Co(2+), which readily adopt different coordination states and geometries, are thus favored. Combining our structures with biochemical data, we identified M2 as the high-affinity metal site. Zn(2+) activates in the M1 site, where octahedral geometry is not essential for catalysis, but inhibits in the M2 site, because it is unable to assume octahedral geometry but remains trigonal bipyramidal. Finally, we propose that Lys205-Gln81-Gln80 form a hydrophilic channel to speed product release from the active site.     

Coordination geometries of metal ions in d- or l-captopril-inhibited metallo-beta-lactamases

d- and l-captopril are competitive inhibitors of metallo-beta-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of gamma-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II)1-BcII, the thiolate sulfur of d-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II)1 and Co(II)2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II)1-BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II)1-CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for d-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-beta-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.     

Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors

Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At about 30 nM IN (20 min at 37 degrees C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of about 70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (about 125 microg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.     

Antibody-based sensors for heavy metal ions

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA™ 3000. Four different monoclonal antibodies specific for complexes of EDTA–Cd(II), DTPA–Co(II), 2,9-dicarboxyl-1,10-phenanthroline–U(VI), or cyclohexyl–DTPA–Pb(II) were incubated with the appropriate soluble metal–chelate complex. In the microwell assay format, the immobilized version of the metal–chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10–1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal–chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25±11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81–7.77% and 3.62–14.16% for within run and between run precision, respectively.     

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.     

Insulin release has no absolute requirement for extracellular monovalent ions

Current chemiosmotic models of exocytosis ascribe an essential role to the influx of extracellular monovalent anions or cations into the secretory granules apposed to the plasma membrane. These hypotheses were tested by measuring insulin release in sucrose media devoid of monovalent ions. A small response to glucose (25% of controls) was still observed, which could be potentiated by isobutylmethylxanthine and suppressed by cobalt or low temperature. Substitution of Ba2+ for Ca2+ triggered a practically normal release of insulin that was inhibited by blockers of Ca2+ channels (cobalt or D 600) and abolished by low temperature. These results show that insulin release remains possible in the absence of extracellular monovalent ions and, therefore, that the chemiosmotic models of exocytosis do not entirely apply to insulin release.     

Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-coAs and requirement for GTP or ATP and Mg(2+) in addition to its known hydroxylation cofactors

Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs. A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E. coli and subsequently purified as an active protein. The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate. The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3-methylhexadecanoic acid. Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated. Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively. Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4-methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain. In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors. Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl-CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway.     

Pituitary hormone releasing or inhibiting activity of metal ions present in hypothalamic extracts

In vitro trophin-releasing and inhibiting activities of certain purified fractions from bovine hypothalamus were apparently due primarily to the presence of divalent metals. The most abundant metal was copper which at concentrations less than 1 μg/ml markedly stimulated the release of all hormones. Zinc ion which was much less potent than copper, inhibited release of prolactin but stimulated release of the other hormones. Nickel, as low as 1 μg/ml, specifically inhibited prolcatin release; at much higher levels it enhanced release of the other hormones.     

Copper chaperones: personal escorts for metal ions

Copper serves as the essential cofactor for a number of enzymes involved in redox chemistry and virtually all organisms must accumulate trace levels of copper in order to survive. However, this metal can also be toxic and a number of effective methods for sequestering and detoxifying copper prevent the metal from freely circulating inside a cell. Copper metalloenzymes are therefore faced with the challenge of acquiring their precious metal cofactor in the absence of available copper. To overcome this dilemma, all eukaryotic organisms have evolved with a family of intracellular copper binding proteins that help reserve a bioavailable pool of copper for the metalloenzymes, escort the metal to appropriate targets, and directly transfer the copper ion. These proteins have been collectively called copper chaperones. The identification of such molecules has been made possible through molecular genetic studies in the bakers' yeast Saccharomyces cerevisiae. In this review, we highlight the findings that led to a new paradigm of intracellular trafficking of copper involving the action of copper chaperones. In particular, emphasis will be placed on the ATX1 and CCS copper chaperones that act to deliver copper to the secretory pathway and to Cu/Zn superoxide dismutase in the cytosol, respectively.     

Mutant forms of beta-galactosidase with an altered requirement for magnesium ions.

By random approaches we have previously isolated many variants of Escherichia coli beta-galactosidase within a short contiguous tract near the N-terminus (residues 8-12 of wild-type enzyme), some of which have increased stability towards heat and denaturants. The activity of these mutants was originally analysed and quantitated in situ in activity gels without the addition of magnesium ions to the buffer system. We now show that the improved stability is only observable under such conditions of limiting magnesium ion concentrations or in the presence of appropriate concentrations of a metal chelator. In the presence of EDTA, purified preparations of one of these mutant enzymes were much more resistant to denaturants than wild-type, but this differential was completely nullified in the presence of 1 mM Mg2+. However, the stability of this mutant enzyme in EDTA was lower than that shown by it, or the wild-type enzyme, in the presence of magnesium ions. In addition, certain alterations within another N-terminal tract (residues 27-31 of wild-type) resulted in enzymes with greater dependence on Mg2+ than natural beta-galactosidase. We conclude that a small number of residue changes in a large protein can profoundly modulate the requirement for metal ion stabilization, allowing partial abrogation of this need in certain cases. Thus, some enzymes which require divalent metal ions for structural purposes only may be engineered towards metal independence.     

Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates

Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP) 相似文献   

Equilibrium sorption isotherm for metal ions on tree fern

A new sorbent system for removing heavy metal ions, such as Zn(II), Cu(II) and Pb(II), from aqueous solutions has been investigated. This new sorbent is tree fern, an agriculture product. Variables of the system include solution temperature and sorbent particle size. The experimental results were fitted to the Langmuir, Freundlich and Redlich–Peterson isotherms to obtain the characteristic parameters of each model. Both the Langmuir and Redlich–Peterson isotherms were found to well represent the measured sorption data. According to the evaluation using the Langmuir equation, the maximum sorption capacities of metal ions onto tree fern were 7.58 mg/g for Zn(II), 10.6 mg/g for Cu(II) and 39.8 mg/g for Pb(II). It was noted that an increase in temperature resulted in a higher metal loading per unit weight of the sorbent. Decreasing the particle sizes of tree fern led in an increase in the metal uptake per unit weight of the sorbent.