Cadmium, zinc and copper biosorption mediated by Pseudomonas veronii 2E

Adsorption properties of bacterial biomass were tested for Cd removal from liquid effluents. Experimental conditions (pH, time, cellular mass, volume, metal concentration) were studied to develop an efficient biosorption process with free or immobilised cells of Pseudomonas veronii 2E. Surface fixation was chosen to immobilise cells on inert surfaces including teflon membranes, silicone rubber and polyurethane foam. Biosorption experiments were carried out at 32 °C and controlled pH; maximal Cd(II) retention was observed at pH 7.5. The isotherm followed the Langmuir model (K_d = 0.17 mM and q_max = 0.48 mmol/g cell dry weight). Small changes in the surface negative charge of cells were observed by electrophoretic mobility experiments in presence of Cd(II). In addition, biosorption of 40% Cu(II) (pH 5 and 6.2) and 50% Zn(II) and 50% Cd(II) (pH 7.5) was observed from mixtures of Cu(II), Zn(II) and Cd(II) 0.5 mM each.     

Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient

A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.     

Partial purification and characterization of the components of the neutral ribonuclease II-inhibitor system of normal and distrophic mouse skeletal muscle

The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.127.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in (a) purification behavior, (b) apparent molecular weights of approximately 50 000, (c) thermal denaturation (50% loss of activity in 5 min at 73.5 degrees C), (d) isoelectric points (pH 4.8), and (e) procedures for reversible resolution into free inhibitor and free RNase II. The free RNase II species are also similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent molecular weights of 11 500 compared with 13 700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95 degrees C. The active RNase II prepared directly from muscle, by resolution of inhibitor complexes or by organic mercurial treatment of the inhibitor complexes, all have identical pH-activity profiles in 200 mM KC1 with an optimum near pH 7.0. In comparison RNase A has an optimum pH near 7.5 and its activity decreases more rapidly as KC1 concentration is increased above 50 mM KC1. RNase II inhibitor obtained by resolution of the purified complexes or by direct isolation in the free form from normal muscle extracts has an apparent molecular weight of 42 000 and is very sensitive to heat; it loses all activity at 40 degrees C in 5 min. These studies (a) provide methods for obtaining useful amounts of the components of the neutral RNase II - inhibitor system from muscle, (b) provide the first method reported for the reversible resolution of RNase II - inhibitor complexes, (c) fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice, (d) establish interesting differences between the apparently homologous enzymes, murine muscle neutral RNase II, and bovine pancreatic RNase A, and (e) provide a substantially lower molecular weight estimate for RNase II inhibitor from muscle than has been reported for the inhibitor from liver, kidney, and placenta.     

Evidence for cadmium uptake through Nramp2: metal speciation studies with Caco-2 cells.

The specific uptake of 0.3 microM (109)Cd by the TC7 clone of the human enterocytic-like Caco-2 cells increased 4-fold as the pH(out) was lowered from 7.5 to 5.5; the stimulatory effect of acidic media being more pronounced when the level of the free ion (109)Cd(2+), relative to total (109)Cd, was increased. The initial uptake rate was 12-fold higher under conditions, optimizing (109)Cd(2+) accumulation over that of (109)CdCl(2-n)(n) (NO(-)(3)/pH(out) 5.5); a saturable system of transport has been characterized (K(m) = 1.1 +/- 0.1 microM, V(max) = 87 +/- 3 pmol/3 min/mg protein). An excess of Fe(2+) failed to affect (109)Cd uptake when the pH(out) was 7.4, whereas a strong inhibition was observed under NO(-)(3)/pH(out) 5.5 conditions. In contrast, the maximal inhibitory effect of Zn(2+) was observed under Cl(-)/pH(out) 7.4 conditions. This results strongly suggest that Fe(2+) may compete with Cd(2+) for Nramp2, whereas Zn and CdCl(2-n)(n) compete for another system of transport that has yet to be identified.     

The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.     

Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein

The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells. Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values. At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5). To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution. The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding. A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface. Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces. Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed.     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

Phytic acid-enhanced metal ion exchange reactions: the effect on carboxypeptidase A

On the basis of the known interaction of phytic acid to form soluble or insoluble complexes with cations, the effect of this naturally occurring polydentate ligand on carboxypeptidase A, a zinc-containing metalloenzyme, and its Co(II)-substituted derivative, has been studied. Under conditions of rigorous exclusion of adventitious metal ions, phytate showed no inhibitory effect. However, the addition of Cu(II) ions to form soluble phytate-Cu(II) complexes at pH 7.2 and 25 degrees C caused more than a 95% decrease in activity. The Cd(II) ion was nearly as effective but other ions showed only a small or no effect. In the absence of phytate, incubation of the enzyme with Cu(II) or Cd(II) at the same concentration produced only about a 25% reduction in activity. The decrease in activity followed first-order kinetics, and the rate constant was the same (1.2 x 10(-4) sec-1) as seen upon incubation with EDTA. However, in contrast to that observed upon incubation of the enzyme with phytate and Cu(II), exposure to EDTA produced a complete loss in activity which could be regained by addition of Zn(II) to the assay solution. In the former case, not only was there residual activity left after incubation at pH 7.2 for 24 hrs at 25 degrees C, but the initial activity could not be regained under similar assay treatment. An increase in either the Cu(II) or phytate concentration while the other was kept constant, yielded saturation curves with maximal effect at 3 x 10(-5) M for Cu(II) and at 5 x 10(-5) M for phytate (enzyme at ca. 10(-6) M). At these ratios, all of the cupric ions are completely bound to phytate as determined by ion-selective potentiometry. A preparative scale reaction of phytate and Cu(II) with carboxypeptidase A (kcat 8460 min-1; K'M 0.23 mM with CBZ-glycyl-glycyl-L-phenylalanine as substrate at pH 7.5, 25 degrees C) gave a product isolated in 95% yield but with lower activity (kcat 198 min-1; K'M 0.25 mM). A Cu(II)-carboxypeptidase preparation had similar kinetic parameters (kcat 207 min-1; K'M 0.34 mM). This near identity of constants suggested that a metal exchange reaction had occurred, i.e., incubation of Zn(II)-carboxypeptidase with a phytate-Cu(II) complex resulted in not only the removal of the zinc ion from the active site but also the sequential and rapid incorporation of a cupric ion into the apoenzyme so formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Active site specific cadmium(II)-substituted horse liver alcohol dehydrogenase: crystal structures of the free enzyme, its binary complex with NADH, and the ternary complex with NADH and bound p-bromobenzyl alcohol

Three crystal structures have been determined of active site specific substituted Cd(II) horse liver alcohol dehydrogenase and its complexes. Intensities were collected for the free, orthorhombic enzyme to 2.4-A resolution and for a triclinic binary complex with NADH to 2.7-A resolution. A ternary complex was crystallized from an equilibrium mixture of NAD+ and p-bromobenzyl alcohol. The microspectrophotometric analysis of these single crystals showed the protein-bound coenzyme to be largely NADH, which proves the complex to consist of CdII-LADH, NADH, and p-bromobenzyl alcohol. Intensity data for this abortive ternary complex were collected to 2.9-A resolution. The coordination geometry in the free Cd(II)-substituted enzyme is highly similar to that of the native enzyme. Cd(II) is bound to Cys-46, Cys-174, His-67, and a water molecule in a distorted tetrahedral geometry. Binding of coenzymes induces a conformational change similar to that in the native enzyme. The interactions between the coenzyme and the protein in the binary and ternary complexes are highly similar to those in the native ternary complexes. The substrate binds directly to the cadmium ion in a distorted tetrahedral geometry. No large, significant structural changes compared to the native ternary complex with coenzyme and p-bromobenzyl alcohol were found. The implications of these results for the use of active site specific Cd(II)-substituted horse liver alcohol dehydrogenase as a model system for the native enzyme are discussed.     

Chloride binding to alkaline phosphatase. 113Cd and 35Cl NMR

Chloride binding to alkaline phosphatase from Escherichia coli has been monitored by 35Cl NMR for the native zinc enzyme and by 113Cd NMR for two Cd(II)-substituted species, phosphorylated Cd(II)6 alkaline phosphatase and unphosphorylated Cd(II)2 alkaline phosphatase. Of the three metal binding sites per enzyme monomer, A, B, and C, only the NMR signal of 113Cd(II) at the A sites shows sensitivity to the presence of Cl-, suggesting that Cl- coordination occurs at the A site metal ion. From the differences in the chemical shift changes produced in the A site 113Cd resonance for the covalent (E-P) form of the enzyme versus the noncovalent (E . P) form of the enzyme, it is concluded that the A site metal ion can assume a five-coordinate form. The E-P form of the enzyme has three histidyl nitrogens as ligands from the protein to the A site metal ion plus either two water molecules or two Cl- ions as additional monodentate ligands. In the E . P form, there is a phosphate oxygen as a monodentate ligand and either a water molecule or a Cl- ion as the additional monodentate ligand. The shifts of the 113Cd NMR signals of the unphosphorylated Cd(II)2 enzyme induced by Cl- are very similar to those induced in the E-P derivative of the same enzyme, supporting the conclusion that the phosphoseryl residue is not directly coordinated to any of the metal ions. Specific broadening of the 35Cl resonance from bulk Cl- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggesting an influence by occupancy of the B site on the interaction of monodentate ligands at the A site. A reduction in this quadrupolar broadening is observed upon phosphate binding at pH values where E . P is formed, but not at pH values where E-P is the major species, confirming a specific interaction of Cl- at the A site, the site to which phosphate is bound in E . P, but not in E-P. For the zinc enzyme, a significant decrease in phosphate binding affinity can be shown to occur at pH 8 where one monomer has a higher affinity than the other.     

Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.     

A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

A soluble F(1)-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C when kept in 20% glycerol. Soluble F(1)-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) =6.58, pK(2)=7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1)=6.42, pK(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F(1)-ATPase with I(50) of 18.6 ng/ml and 2.2 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM NBD-Cl). These data suggest a conformational change transmission between F(0) and F(1) sectors and slight conformational differences between soluble F(1) and membrane-bound F(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Effects of pH and Ca2+ on monomer-dimer and monomer-tetramer equilibria of chromogranin A.

Chromogranin A is a high capacity, low affinity Ca2+ binding protein which undergoes Ca2+- and pH-dependent conformational changes, and has recently been suggested to play a Ca2+-buffering role in the secretory vesicle of adrenal medullary chromaffin cell, the major inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store of chromaffin cell (Yoo, S.H., and Albanesi, J.P. (1990) J. Biol. Chem. 265, 13446-13448). In the present study, it is shown that chromogranin A exists in a monomer-dimer equilibrium at pH 7.5 and in a monomer-tetramer equilibrium at pH 5.5. The pH appears monomer-tetramer equilibrium at pH 5.5. The pH appears to be a necessary and sufficient factor determining the types of oligomers formed. Although Ca2+ did not change the type of oligomerization, it had a very significant effect on the values of the thermodynamic parameters characterizing the associations. The delta G0 values for a monomer-dimer equilibrium were -7 to -8 kcal/mol, while those for a monomer-tetramer equilibrium were -20 to -23 kcal/mol. At pH 5.5, the values of delta H0, delta S0, and delta C0p were large and negative in the absence of Ca2+ and large and positive in the presence of 35 mM Ca2+, implying markedly different reaction mechanisms. Extrapolation of the results to 37 degrees C and 1 mM chromogranin A suggests that chromogranin A is virtually 100% tetramer at pH 5.5 in the presence of 35 mM Ca2+ but is 96% dimer at pH 7.5 in the absence of Ca2+, the two conditions resembling those seen in vivo. These results suggest that chromogranin A is mostly dimer in the endoplasmic reticulum and cis-Golgi area and is essentially all tetramer in the vesicle.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.     

The effect of pH and various cations on the GTP hydrolysis of rice heterotrimeric G-protein alpha subunit expressed in Escherichia coli

Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997). The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the MgCl(2) concentration. The steady state rate constant (k(cat) ) for GTP hydrolysis of RGA1 at 2 mM MgCl(2) was 0.0075 +/- 0.0001 min(-1). Here, we examined the effects of pH and cations on the GTPase activity. The optimum pH at 2 mM MgCl(2) was approximately 6.0; whereas, the pH at 2 mM NH(4)Cl was approximately 4.0. The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate (k(cat)= 0.0353 min(-1)) under the condition of 2 mM NH(4)Cl at pH 4.0 was the highest. It corresponded to about 3.24-fold of the k(cat) value of 0.0109 min(-1) in the presence of 2 mM MgCl(2) at pH 6.0.     

Investigation of pH-dependent DNA-metal ion interactions by surface plasmon resonance

Ni(II) and Zn(II) M-DNA formation and denaturation of double-stranded DNA (dsDNA) by Cd(2+) were monitored by surface plasmon resonance (SPR). When exposed to immobilized 30 bp 50% GC dsDNA, Zn(2+) and Ni(2+) were found to give signals indicative of a conformational change at pH 8.5 but not 7.5, while Mg(2+) and Ca(2+) caused small changes at both pHs. The concentrations that gave 50% of the maximum responses were 0.06 and 0.50 mM for Zn(2+) and Ni(2+), respectively. At pH 8.5, Cd(2+) denatured over 40% of the dsDNA, while other metals denatured less than 5% of the DNA. Smaller pH-dependent signals were induced by Zn(2+), Ni(2+) or Cd(2+) with 50% GC single-stranded DNA (ssDNA), and with a homopolymer of d(T)30. Homopolymers d(A)30 and d(C)30 showed small signals that were largely independent of pH in the presence of Zn(2+) or Ni(2+).     

Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: spectral evidence for allosteric regulation by pH and bicarbonate ion

Cobalt(II)-substituted Haemophilus influenzae β-carbonic anhydrase (HICA) has been produced by overexpression in minimal media supplemented with CoCl(2), enabling kinetic, structural, and spectroscopic characterization. Co(II)-substituted HICA (Co-HICA) has comparable catalytic activity to that of wild-type enzyme with k(cat)=82±19 ms(-1) (120% of wild-type). The X-ray crystal structure of Co-HICA was determined to 2.5? resolution, and is similar to the zinc enzyme. The absorption spectrum of Co-HICA is consistent with four-coordinate geometry. pH-dependent changes in the absorption spectrum of Co-HICA, including an increase in molar absorptivity and a red shift of a 580 nm peak with decreasing pH, correlate with the pH dependence of k(cat)/K(m). The absence of isosbestic points in the pH-dependent absorption spectra suggest that more than two absorbing species are present. The addition of bicarbonate ion at pH 8.0 triggers spectral changes in the metal coordination sphere that mimic that of lowering pH, supporting its hypothesized role as an allosteric inhibitor of HICA. Homogeneously (99±1% Co) and heterogeneously (52±5% Co) substituted Co-HICA have distinctly different colors and absorption spectra, suggesting that the metal ions in the active sites in the allosteric dimer of Co-HICA engage in intersubunit communication.     

Interaction and inhibitory effect of ammonium cation in the oxygen evolving center of photosystem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.