Isolation and biochemical characterization of maleic-acid hydratase, an iron-requiring hydro-lyase

A procedure for the isolation of maleic acid hydratase (D-malate hydro-lyase, EC 4.2.1.31) of about 95% purity from rabbit kidneys is described. The enzyme consists of a single polypeptide chain of 582 amino-acid residues with an approximate molecular mass of 68 kDa. The enzyme is very unstable and has an absolute requirement for chloride ions. Addition of sodium sulphide during the purification process was essential to maintain the enzyme in an activatable state. The pure preparation has low activity but responds to activation with Fe2+ ions, Na2S and a thiol. The sequence of adding the activating reagents is critical to achieve optimal activity. Ni2+ and to a lesser extent Co2+ can replace iron in the activation process. The enzyme incorporates 4-5 mol iron/mol and 4.5-6 mol sulphide/mol during activation. In this process an [Fe-S] cluster appears to be built up, as indicated by optical and electron paramagnetic resonance (EPR) spectroscopy. In activated samples exposed to air the [Fe-S] cluster is EPR-detectable through an axial signal with g = 2.01 and g = 2.029 whose temperature and power saturation characteristics were similar to those of other [3Fe-xS] clusters. The activated enzyme, however, is readily inactivated even upon minor manipulation with destruction of the iron-sulfur core.     

红球菌低分子量型腈水合酶的异源激活及激活子的结构域功能

腈水合酶激活子具有亚基自身交换伴随子或者金属离子伴随子的功能,能够辅助腈水合酶摄取金属离子,对于腈水合酶的活性表达必不可少。与腈水合酶自身相比,激活子的序列保守性低,研究其激活作用的特点,探索其结构与功能之间的关系,对于理解腈水合酶的成熟机制具有重要意义。将红球菌Rhodococcus rhodochrous J1低分子量型腈水合酶L-NHase分别与4种异源激活子组合共表达,测定异源激活子对L-NHase的激活作用,进一步对激活子进行序列分析和结构模拟,并研究关键结构域的功能。结果表明,4种异源激活子均能激活L-NHase,但激活后L-NHase的比酶活存在差异,激活子A对L-NHase的激活程度最高,激活后的L-NHase比酶活为出发酶的97.79%;激活子G对L-NHase的激活程度最低,激活后的L-NHase比酶活为出发酶的23.94%。激活子E和激活子G具有保守结构域TIGR03889,缺失其中部分序列会使两者的激活作用基本丧失。将激活子G的N端序列替换为激活子E的N端序列,并将激活子E的C端序列添加至激活子G的C端,能够使L-NHase的比酶活提高178.40%。激活子的激活作用具有普遍性和特异性,其保守结构域对激活作用至关重要,同时N端结构域和C端结构域也对激活作用产生重要影响。     

Metal ion requirement of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase from rat liver

The metal ion requirement for both enzymatic activitiesof the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosaminekinase (E.C. 5.1.3.14/ 2.7.1.60), the key enzyme of N-acetylneuraminic acidbiosynthesis in ratliver, was investigated. UDP-N-acetylglucosamine 2-epimerase was active inimida-zole/HCl buffer in the complete absence of any metal ion. 200 mM Na + , K + , Rb + and Cs +activated enzymeactivity up to five-fold, whereas lower concentrations of thesemonovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimeraseactivity. In sodium phosphate buffer the enzyme activitywas increased by 0.5 mM Mg , Sr , Ba and Mn , while in the presence of 200 mM NaCl UDP-N-acetyl-glucosamine2-epimerase activity showed astronger activation by these divalent metal ions. In imidazole/HClbuffer, UDP-N-acetylglucosamine2-epimerase activity was partially inhibited by 0.5 mM Be , Mg , Ba ,Mn , Sn and Fe , and completely inhibited by 0.5 mM Zn and Cd . Divalent metal ions were essen-tialforN-acetylmannosamine kinase activity, the most effective being Mg , followed byMn and Co .The optimal concentration of these metal ions was 3 mM. Less effective were Ni and Cd , whereas Ca ,Ba , Cu , Fe and Zn showed no effect on enzyme activity.     

Kinetic and spectroscopic studies on the quercetin 2,3-dioxygenase from Bacillus subtilis

Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin and molecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, the only known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe2+-dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme in Escherichia coli grown on minimal media in the presence of a number of divalent metals. The addition of Mn2+, Co2+, and Cu2+ generated active enzymes, but the addition of Zn2+, Fe2+, and Cd2+ did not increase quercetinase activity to any significant level over a control in which no divalent ions were added to the media. The Mn2+- and Co2+-containing QueDs were purified, characterized by metal analysis and EPR spectroscopy, and studied by steady-state kinetics. Mn2+ was found to be incorporated nearly stoichiometrically to the two cupin motifs. The hyperfine coupling constant of the g = 2 signal in the EPR spectra of the Mn2+-containing enzyme showed that the two Mn2+ ions are ligated in an octahedral coordination. The turnover number of this enzyme was found to be in the order of 25 s(-1), nearly 40-fold higher than that of the Fe2+-containing enzyme and similar in magnitude to that of the Cu2+-containing quercertin 2,3-dioxygenase from Aspergillus japonicus. In addition, kinetic and spectroscopic data suggest that the catalytic mechanism of QueD is different from that of the Aspergillus quercetinases but similar to that proposed for the extradiol catechol dioxygenases. This study provides evidence that Mn2+ might be the preferred cofactor for this enzyme and identifies QueD as a new member of the manganese dioxygenase family.     

Requirement of an essential thiol group and ferric iron for the activity of the progesterone-induced porcine uterine purple phosphatase.

The progesterone-induced purple phosphatase isolated from the uterine flushings of pigs is activated by a variety of reagents that cleave disulfide bonds, including 2-mercaptoethanol, dithiothreitol, L-ascorbate, L-cysteine, sulfite, and cyanide. It is inhibited by various mercurials, iodoacetamide, O-iodosobenzoate, and hydrogen peroxide. Thiols increase the specific phosphatase activity from 25 to about 300 units per mg of enzyme. This activation is accompanied by a shift in the extinction maximum to higher energy to yield a protein with a pink coloration. Following maximum activation there is a gradual decrease in enzyme activity and protein color which is accompanied by loss of ferrous iron from the protein. Sodium dithionite at 10 mM or higher causes an immediate inhibition of phosphatase activity and bleaching of color, and can be used to prepare the iron-free apoprotein. The latter can be partially reactivated by Fe3+ salts but not by Fe2+. The Fe3+ restores the pink form of the enzyme with a specific activity of about 200 units/mg of protein. Cu2+ also causes some reactivation, but other metal ions were ineffective. ESR studies showed that the pink form of phosphatase contains approximately 1 atom of high spin ferric iron per molecule. It is concluded that the phosphatase requires a free thiol and Fe3+ for activity. Reduction of the iron leads to complete loss of both color and enzyme activity. The color change from purple to pink represents disulfide reduction and is not due to reduction of iron.     

Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.     

Iron-containing acid phosphatases: Characterization of the metal-ion binding site of the enzyme from pig allantoic fluid

All of the iron can be removed from the violet acid phosphatase of pig allantoic fluid by treatment with sodium dithionite at pH 4.9. Of the two moles of iron present per mole of enzyme (40,000 g), half is lost rapidly, and the remainder much more slowly. Removal of half of the iron causes complete loss of acid phosphatase activity. Conditions have been defined for the isolation and complete reconstitution [by Fe(II) and β-mercaptoethanol] of two apoenzymes, designated “iron-free” and “one-iron” apoenzymes. Zn²⁺ ions restore most of the acid phosphatase activity to the one-iron apoenzyme but not to the iron-free enzyme. No metal ions other than Fe(II) and Fe(III) restore significant activity to the iron-free apoenzyme, but Zn(II) and Ni(II) bind tightly to it.     

Spectroscopic studies of isopenicillin N synthase. A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. M?ssbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.     

Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation.

The characteristics of hydroperoxide activation of 5-lipoxygenase were examined in the high speed supernatant fraction prepared from rat polymorphonuclear leukocytes. Stimulation of 5-lipoxygenase activity by the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) reaction product was strongly dependent on the presence of thiol compounds. Various reducing agents such as mercaptoethanol and glutathione (0.5-2 mM) inhibited the reaction and increased the concentrations of 5-HPETE (1-10 microM) necessary to achieve maximal arachidonic acid oxidation. The requirement for 5-HPETE was not specific and could be replaced by H2O2 (10 microM) but not by the 5-hydroxyeicosatetraenoic acid (5-HETE) analogue. Furthermore, gel filtration chromatography of the soluble extract from leukocytes resolved different fractions which can increase the hydroperoxide dependence or fully replace the stimulation by 5-HPETE. Maximal activity of the 5-HPETE-stimulated reaction required Ca2+ ions (0.2-1 mM) and ATP with the elimination of the HPETE requirement at high ATP concentrations (2-4 mM). In addition, NADPH (1-2 mM), FAD (1 mM), Fe2+ ions (20-100 microM) and chelated Fe3+ (0.1 mM-EDTA/0.1 mM-FeCl3) all markedly increased product formation by 5-lipoxygenase whereas NADH (1 mM) was inhibitory and Fe3+ (20-100 microM) alone had no effect on the reaction. The stimulation by Fe2+ ions and NADPH was also observed under various conditions which increase the hydroperoxide dependence such as pretreatment of the enzyme preparation with glutathione peroxidase or chemical reduction with 0.015% NaBH4. These results provide evidence for an hydroperoxide activation of 5-lipoxygenase which is not product-specific and is modulated by thiol levels and several soluble components of the leukocytes. They also indicate that stimulation of 5-lipoxygenase activity can contribute to increase lipid peroxidation in iron and nucleotide-promoted reactions.     

Cloning and characterization of chironomidae ferrochelatase: Copper activation of the purified ferrochelatase

All organisms utilize ferrochelatase (EC 4.99.1.1) to catalyze the insertion of ferrous iron into protoposphyrin IX in the terminal step of the heme biosynthetic pathway. Different metal-binding affinity for the enzyme leads to changes in enzyme activity. In this work, we have cloned and over-expressed the enzyme from chironomidae in E. coli. The enzyme was purified and characterized. The recombinant enzyme showed higher enzymatic activity (four-fold increase) in the presence of copper ions and unaffected by calcium ions. Other divalent metal ions including magnesium, manganese, lead, reduced the enzyme activity by >60%. Over 90% of the enzyme activity was inhibited by Zn2+. The sequence alignment of amino acid residues reveals 83% homology with other ferrochelatases. The results of electron proton resonance (EPR) analysis showed that Fe2+ ion was present in the cluster of the recombinant enzyme complex. The recombinant enzyme also contained the [2Fe-2S] center with two-fold higher enzymatic activity than human ferrochelatase.     

Occurrence of 3-hydroxyretinol in the freshwater fish Bagarius bagarius and Wallago attu. Isolation and synthesis.

1. Four different types of alpha-mannosidase activity were shown to occur in several tissues from the rat. There is the Zn2+-dependent enzyme, active at acidic pH, and three enzymes that are active near to neutral pH. 2. The 'neutral' enzymes are activated by Fe2+, Co2+ or Mn2+. 3. Optimum activities for these three enzymes are shown at pH values of 5.2, 6.5 and 7.3. The activity at pH6.5 is the only one evident without metal-ion activation, but activity is enhanced by all three metal ions. The activity at pH 5.2 is seen only in the presence of Fe2+ or Co2+, and the activity at pH7.3 is seen only in the presence of Co2+ or Mn2+ and in a non-chelating buffer medium. 4. The pH6.5-active enzyme is inactivated by EDTA, but activity is restored by excess of metal ion. 5. The enzymes differ markedly in their stability. The pH6.5-active enzyme is very labile and the pH7.3-active enzyme is the most stable. 6. Tissue preparations vary widely in their activity at pH6.5, but where activity is low it can be increased by incubation with one of the activating metal cations. 7. All the enzymes active at neutral pH are inhibited by heavy-metal ions and stabilized to some extent by thiol groups.     

Purification and characterization of two types of fumarase from Escherichia coli

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.     

Distinctive iron requirement of tryptophan 5-monooxygenase: TPH1 requires dissociable ferrous iron

A peripheral type of tryptophan 5-monooxygenase (EC 1.14.16.4), TPH1, is very unstable in vitro, but the inactivation was reversible and full reactivation occurs upon anaerobic incubation with a high concentration of dithiothreitol (DTT, 15 mM). In this study, distinctive iron requirement of TPH1 was revealed through analysis of the enzyme's inactivation and activation by DTT. For this purpose, all the glasswares, plastics, Sephadex G-25 gels, and reagents including protein solutions had been treated with metal chelators, and apo-TPH was prepared by treatment with EDTA. Apo-TPH thus prepared exclusively required free Fe2+ for its catalytic activity; 10(-8) M was enough under the strict absence of Fe3+ but 10(-12) M was too low. No other metal ions including Fe3+ were effective. It appeared that Fe3+ bound to the enzyme with a higher affinity than Fe2+, resulting in the inactivation. Ascorbate, a non-thiol reducing agent, did not substitute DTT in the activation of TPH1, but enhanced the Fe2+-dependent activity of apo-TPH as effectively as DTT. Thus, the DTT-activation was essentially substituted by preparation of apo-TPH by the EDTA treatment and the assay of apo-TPH in the presence of Fe2+ and ascorbate. The activation of TPH1 by incubation with DTT was accompanied by exposure of 9 sulfhydryls out of the total 10 cysteine residues, but the cleavage of disulfide bonds seemed not to be crucial, even if it occurred. The effect of DTT was substituted by some other sulfhydryls whose structure was analogous to that of commonly used metal chelators. Based on these observations, the following dual roles of DTT are proposed: (1) in the activation of TPH, DTT removes inappropriate bound iron (Fe3+) as a chelator, keeping Fe3+ away from the enzyme's binding site which needs to bind Fe2+ for the catalytic activity, and (2) in both the activation and reaction processes, DTT prevents oxidation of Fe2+ to Fe3+ as a reducing agent.     

Pseudomonas aeruginosa acid phosphatase. Activation by divalent cations and inhibition by aluminium ion.

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.     

Mn2+ is a native metal ion activator for bacteriophage lambda protein phosphatase

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP. Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear [(Mn(2+))(2)] species bound to lambdaPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM [(Mn(2+))(2)]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn(2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.     

The effect of substrate, dihydrobiopterin, and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4)) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (E(m)(Fe(III)/Fe(II))) were studied by EPR spectroscopy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E(m) = +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH(4) (E(m) (quinonoid-BH(2)/BH(4)) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme decreased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrobiopterin (BH(2)), the stable oxidized form of BH(4), and of dopamine. All three ligands induced a decrease in the E(m) value of the iron to +123 +/- 4 mV (L-Phe), +110 +/- 20 mV (BH(2)), and -8 +/- 9 mV (dopamine). On the basis of these data we have calculated that the binding affinities of L-Phe, BH(2), and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly, an E(m) value comparable with that of the ligand-free, resting form of wt-hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of both L-Phe and BH(2), representing an inactive model for the iron environment under turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the reductive activation of the enzyme and the catalytic mechanism.     

Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.     

Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells

This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry.     

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.     

Purification and characterization of a purple acid phosphatase from rat spleen

An acid phosphatase species which is activated by Fe2+ was purified 3,700-fold from rat spleen by chromatography on columns containing Blue-Sepharose, concanavalin A-Sepharose, Sephadex G-100, and CM-Sephadex. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, phosphoproteins, and thiamine pyrophosphate with Km values of 10(-4) to 10(-3) M at an optimal pH of 5.0-5.8. Co-purification of the acid phosphatase and acid phosphoprotein phosphatase indicated that they were identical. The purified enzyme was glycoprotein in nature, showing four heterogeneous forms on acid polyacrylamide gel electrophoresis (pI values, 7.8, 8.0, 8.3, and 8.5), but it gave a molecular weight of 33,000 on sodium dodecyl sulfate-gel electrophoresis and gel permeation chromatography. The enzyme had a purple color (lambda max 545 nm) and contained 2 iron atoms per enzyme molecule. Among reductants, ascorbic acid and Fe2+ were the best activators, although their combined effect was not additive. Fe2+ and ascorbic acid both changed the purple enzyme into the same active form (lambda max 515 nm), giving almost the same kinetic constants for substrates and for inhibitors such as molybdate, phosphate and fluoride. However, low concentrations of Fe2+, from 0.01 mM to 1.0 mM, immediately and reversibly activated the enzyme, whereas high concentrations of ascorbic acid over 1 mM were required for maximal activation, which was slow and irreversible.