Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Reactivation of the EDTA-treated arginase from rat and calf liver.

1. The anionic calf liver arginase, like the cationic rat liver enzyme, is inactivated by EDTA-treatment. The activity is fully restored by Mn2+. A smaller effect is observed with Cd2+, Ni2+ and Co2+. 2. The EDTA-inactivated calf liver arginase, unlike the rat liver enzyme, does not dissociate into subunits, and its mol.wt. (120 000) is unchanged. 3. The reactivation of rat liver arginase subunits (mol.wt. 30 000) by Ni2+ is accompanied, similarly as in the case of Mn2+, by reassociation to the form of mol.wt. 120 000, i.e. the same as for the native enzyme. 4. It is suggested that Mn2+ in arginase is bound at the active site and at the site responsible for maintenance of the oligomeric structure. In calf liver enzyme this binding site is inaccessible to the chelating agent.     

The role of zinc in Bacillus subtilis cytidine deaminase

Cytidine deaminase (CDA) from Bacillus subtilis is a zinc-containing enzyme responsible for the hydrolytic deamination of cytidine to uridine and 2'-deoxycytidine to 2'-deoxyuridine. Titration of the cysteinyl groups of the enzyme with p-hydroxymercuriphenyl sulfonate (PMPS) resulted in release of one zinc ion per subunit. Addition of EDTA to chelate the zinc and dithiothreitol (DTT) to remove PMPS, followed by removal of the low molecular weight compounds by gel filtration, resulted in an apoenzyme with no enzymatic activity. The apoenzyme was almost fully reactivated by addition of zinc chloride, indicating that the zinc ion played a central role in catalysis, in keeping with what has been observed with Escherichia coli CDA [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., and Carter, C. W. J. (1994) J. Mol. Biol. 235, 635-656]. Addition of Cd(2+) or Co(2+) caused partial reactivation of the apoenzyme. Zinc reconstitution of the apoenzyme was strictly dependent on the presence of reducing agents, suggesting that the zinc-ligating cysteines, when unligated, participated in disulfide bond formation. An enzymatically active isoform of the tetrameric CDA protein, containing an extension of 13 amino acids at the C-terminus of each subunit, was used in conjunction with the wild-type CDA in subunit-subunit dissociation studies to show that the zinc ion does not assist in the thermodynamic refolding of the protein. After treatment with PMPS and EDTA, the enzyme existed as unfolded unassociated subunits. Immediately following DTT addition to remove PMPS, the subunits refolded into a tetrameric structure, independent of the presence of zinc.     

Subunit interactions and immobilised dimers of human liver arginase.

Incubation of soluble human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) with p-hydroxymercuribenzoate resulted in the dissociation of the enzyme into active dimers. Addition of 2-mercaptoethanol resulted in the regeneration of the tetrameric enzyme. When arginase, bound covalently to nylon, was incubated with p-hydroxymercuribenzoate, matrix-bound dimers were obtained. Incubation of these species with 2-mercaptoethanol resulted in stable, unmodified dimers. Based on this dissociation of arginase, a model with D2-symmetry is suggested for this enzyme. The specific activity, the Km value for arginine, pH optimum and the inhibition constants for ornithine and lysine were determined for monomeric, dimeric and tetrameric forms. It is concluded that the behaviour of the active sites of the monomers is not substantially altered by the interaction of these species in the oligomeric molecule.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.     

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.     

Partial purification of alkaline phosphatase from bovine polymorphonuclear neutrophils and some properties

Alkaline phosphatase was purified from bovine polymorphonuclear neutrophils by butanol extraction and a combination of ion exchange, gel filtration and affinity chromatography. The enzyme was partially purified 2300-fold with a 4.7% yield and a sp. act. of 206 units/mg of protein. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a single activity band with the mol. wt of 165,000. The pH optima for the enzyme were 10.0 with p-nitrophenylphosphate and phenylphosphate and were 9.0 when beta-glycerophosphate, AMP and ADP were used. The enzyme was activated by Mg2+, Mn2+, Co2+ and Ni2+ but was inhibited by Zn2+. The enzyme was inhibited by EDTA and the EDTA-inactivated enzyme was reactivated by Mg2+, Mn2+ and Co2+ but not Zn2+.     

Urea cycle of Fasciola gigantica: purification and characterization of arginase

The ornithine-urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase:canavanase ratio (>10). The enzyme exhibited a maximal activity at pH 9.5 and Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine>isoleucine>ornithine>valine>leucine>proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.     

Spatial proximity of two divalent metal ions at the active site of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is shown to require 2 divalent metal ions/enzyme subunit for maximal enzymatic activity. In the absence of substrate, the tetrameric enzyme binds 1 Mn(II) ion/subunit, whereas in the presence of a nucleotide substrate, adenylylimidodiphosphate, or the product pyrophosphate, there are two Mn(II)-binding sites/subunit. Electron paramagnetic resonance spectra of Mn(II) bound to the enzyme reveal a spin exchange interaction between 2 Mn(II) ions in complexes of enzyme and Mn(II) which also contain adenosylmethionine, K+, and either pyrophosphate or imidotriphosphate. Since a spin exchange interaction requires orbital overlap between the 2 ions, the metal ions must be bound close to one another, and they may share a common ligand.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

Manganese is essential for catalytic activity of Escherichia coli agmatinase.

Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.     

Immobilization of pig muscle enolase. Studies on the activity of subunits

The native dimeric form of enolase from pig muscle was immobilized on Sepharose 4B activated with cyanogen bromide. The amount of matrix-bound enolase, its specific activity and kinetic properties depend on the extent of gel activation with CNBr. Only on the Sepharose activated with small quantities of CNBr the amount of protein which remained after treatment with Gdn.HCl was about 50% of the initially bound enolase, indicating that the enzyme was bound covalently to the matrix through a single subunit. The matrix-bound monomers obtained in this way were inactive and were unable to reassociate to dimers on addition of free subunits. The matrix-bound monomers obtained after KBr treatment were inactive but retained the ability to reassociate into active dimers after addition of free subunits. The results indicate that single matrix-bound subunits of pig muscle enolase are enzymatically inactive and dimeric structure is essential for catalytic activity.     

Trypsin digestion of arginase: evidence for a stable conformation manganese directed.

1. Controlled tryptic digestion of native arginase from rat liver suggests that Mn2+ promotes a stable conformation as shown by the following features. 2. An 18-fold increase in the half-life of arginase activity in the presence of Mn2+ is produced. 3. The stability of subunit B of arginase is increased in the presence of Mn2+ as revealed by SDS-PAGE during the time-course of trypsin cleavage. 4. The different digestion products of arginase with and without Mn2+ appearing during the time-course of tryptic treatment. 5. Different activity/bands protein ratio at any time of the tryptic digestion in the incubation mixtures, with and without Mn2+, are apparent.     

Insights into the interaction of human liver arginase with tightly and weakly bound manganese ions by chemical modification and site-directed mutagenesis studies

Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by Mn(2+). The effect was reversed by hydroxylamine and involved a residue with a pK(a) of 6.5+/-0.1. Half activation with Mn(2+) was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in K(m) for arginine or K(i) for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added Mn(2+) and contained <0.1 Mn(2+)/subunit, whereas H101N was half active and contained 1.2+/-0.1 Mn(2+)/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state.     

Manganese-dependent inhibition of human liver arginase by borate

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.     

Purification and characterization of a neutral protease from rat-liver cytosolic fraction

Rat liver cytosol contains a neutral protease which degrades acetylated hemoglobin and some urea-denatured proteins maximally at pH 7.5. The enzyme was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA and o-phenanthroline, the metal-depleted enzyme can be reactivated by Co2+, Zn2+, Mn2+, or Mg2+, and it is not inhibited by reagents specific for carboxyl, seryl, or thiol proteases. The enzyme has an apparent molecular weight of 200,000 as determined on Sephacryl S-200 column chromatography, and electrophoresis in sodium dodecyl sulfate showed 3 protein bands corresponding to the molecular weights of 110,000, 74,000, and 40,000.     

L-threonine dehydrogenase from Escherichia coli. Identification of an active site cysteine residue and metal ion studies

Pure L-threonine dehydrogenase from Escherichia coli is a tetrameric protein (Mr = 148,000) with 6 half-cystine residues/subunit; its catalytic activity as isolated is stimulated 5-10-fold by added Mn2+ or Cd2+. The peptide containing the 1 cysteine/subunit which reacts selectively with iodoacetate, causing complete loss of enzymatic activity, has been isolated and sequenced; this cysteine residue occupies position 38. Neutron activation and atomic absorption analyses of threonine dehydrogenase as isolated in homogeneous form now show that it contains 1 mol of Zn2+/mol of enzyme subunit. Removal of the Zn2+ with 1,10-phenanthroline demonstrates a good correlation between the remaining enzymatic activity and the zinc content. Complete removal of the Zn2+ yields an unstable protein, but the native metal ion can be exchanged by either 65Zn2+, Co2+, or Cd2+ with no change in specific catalytic activity. Mn2+ added to and incubated with the native enzyme, the 65Zn2(+)-, the Co2(+)-, or the Cd2(+)- substituted form of the enzyme stimulates dehydrogenase activity to the same extent. These studies along with previously observed structural homologies further establish threonine dehydrogenase of E. coli as a member of the zinc-containing long chain alcohol/polyol dehydrogenases; it is unique among these enzymes in that its activity is stimulated by Mn2+ or Cd2+.     

Presence and androgen control of an alkaline phosphatase in the nucleus of rat ventral prostate.

The presence of alkaline phosphatase (EC 3.1.3.1) activity has been demonstrated in nuclei of rat ventral prostate. This enzyme activity remained after washing of isolated nuclei with 0.5% Triton X-100; an acid phosphatase initially present with the nuclear fraction was removed by this treatment. The nuclear alkaline phosphatase, examined by utilizing p-nitrophenyl phosphate as substrate, had a pH optimum of 9.5-10.3, and a broad substrate specificity: p-nitrophenyl phosphate greater than phosphothreonine greater than beta-glycerophosphate greater than phosphoserine. The nuclear phosphatase was sensitive to denaturation by heat or urea treatments and was also inhibited by Pi, L-phenylalanine, homoarginine, dithiothreitol, and EDTA. The EDTA-inhibited enzyme was maximally reactivated by Zn2+, although Mg2+, or Ca2+ were also effective at somewhat higher concentrations. Orchiectomy of adult rats resulted in an increase in the nuclear alkaline phosphatase activity (2-3-fold at 24 or 48 h postorchiectomy). A decline in the protein: DNA ratio also occurred following orchiectomy, but the increase in phosphatase specific activity was evident whether expressed per unit of protein or per unit of DNA. Testosterone replacement following orchiectomy abolished the increase in nuclear phosphatase activity. The results suggest that the prostatic nuclear alkaline phosphatase may be involved in events related to inactivation of the prostate nucleus following androgen deprivation.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.