Role of phosphoenolpyruvate carboxylation in Acetobacter xylinum

Glucose-grown cells of Acetobacter xylinum oxidized acetate only when the reaction mixture was supplemented with catalytic quantities of glucose or intermediates of the citrate cycle. Extracts, prepared by sonic treatment, catalyzed the formation of oxalacetate when incubated with phosphoenolpyruvate (PEP) and bicarbonate. Oxalacetate was not formed in the presence of pyruvate plus adenosine triphosphate. The ability to promote carboxylation of PEP was lower in succinate-grown cells than in glucose-grown cells. PEP carboxylase, partially purified from extracts by ammonium sulfate fractionation, catalyzed the stoichiometric formation of oxalacetate and inorganic phosphate from PEP and bicarbonate. The enzyme was not affected by acetyl-coenzyme A or inorganic phosphate. It was inhibited by adenosine diphosphate in a manner competitive with PEP (K(1) = 1.3 mm) and by dicarboxylic acids of the citrate cycle; of these, succinate was the most potent inhibitor. It is suggested that the physiological role of PEP carboxylase in A. xylinum is to affect the net formation of C(4) acids from C(3) precursors, which are essential for the maintainance of the citrate cycle during growth on glucose. The relationship of PEP carboxylase to other enzyme systems metabolizing PEP and oxalacetate in A. xylinum is discussed.     

CO 2 -fixing enzymes in a marine psychophile

A psychrophilic marine Pseudomonas was found to contain phosphoenolpyruvate (PEP) carboxylase and an adenosine triphosphate-linked PEP carboxykinase. Some properties of these CO(2)-fixing enzymes were compared with those homologous enzymes from the terrestrial mesophile Enterobacter cloacae. The PEP carboxylases from both organisms were activated by acetyl-coenzyme A (CoA) and inhibited by l-aspartate. The enzyme from Pseudomonas was less dependent on the presence of the activator, but maximal activation was attained at acetyl-CoA concentrations much lower (50 mum) than those required to saturate the enzyme from E. cloacae. In both cases the main effect of acetyl-CoA was to decrease the K(m) value for PEP. The activity of PEP carboxylase from Pseudomonas was only slightly inhibited by NaCl, KCl, or NH(4)Cl up to 100 mm, whereas the enzyme from E. cloacae was inhibited by about 70% under similar experimental conditions. Both PEP carboxylase and PEP carboxykinase from Pseudomonas showed considerably lower thermal stability than their counterparts from E. cloacae. Our results suggest that the CO(2)-fixing enzymes from a marine Pseudomonas and E. cloacae are similar in nature and regulation, but they differ in properties related to the peculiar conditions of the marine environment.     

Purification and general properties of δ-aminolaevulate dehydratase from Nicotiana tabacum L

1. delta-Aminolaevulate dehydratase (EC 4.2.1.24) was purified 80-fold from tobacco leaves and its properties were studied. 2. The enzyme had optimum pH7.4 in potassium phosphate buffer, K(m)6.25x10(-4)m at 37 degrees and pH7.4, optimum temperature 45 degrees and an activation energy of 11100 cal./mole. 3. The enzyme lost activity when prepared in the absence of cysteine, and this activity was only partly restored by the later addition of thiols. Reagents for thiol groups inactivated the enzyme. 4. Mg(2+) was essential for activity, and EDTA and Fe(2+) were inhibitory; Mn(2+) was an activator or an inhibitor depending on the concentration.     

Purification of the alliin lyase of garlic, Allium sativum L

1. Alliin lyase (EC 4.4.1.4) was purified up to sevenfold from garlic-bulb homogenates. The enzyme was unstable to storage at -10 degrees , particularly in dilute concentrations, but the addition of glycerol (final concentration 10%, v/v) stabilized the activity completely for at least 30 days. 2. The purified enzyme had an optimum pH for activity at 6.5. The addition of pyridoxal phosphate stimulated the reaction rate and the stimulation became more marked as the purification proceeded. 3. Hydroxylamine (10mum) and cysteine (0.5mm) inhibited the enzyme activity by more than 80%. Spectral studies indicated that cysteine reacted with pyridoxal phosphate bound to the protein. 4. The K(m) values for S-methyl-, S-ethyl-, S-propyl-, S-butyl- and S-allyl-l-cysteine sulphoxides were determined. With S-allyl-l-cysteine sulphoxide the K(m) was 6mm and the V(max.) was greater than those with the other substrates tested. 5. The thioether analogues of the substrates were competitive inhibitors for the lyase reaction. The K(i) decreased with increasing chain length of the alkyl substituent. With S-ethyl-l-cysteine sulphoxide as substrate the K(i) was 33, 8 and 5mm respectively for S-methyl-, S-ethyl- and S-propyl-l-cysteine. 6. The addition of EDTA or Mg(2+), Mn(2+), Co(2+) or Fe(2+) stimulated the reaction rate. Other bivalent cations either had no effect or gave a strong inhibition. In the presence of EDTA no further increase of activity was observed with added Mg(2+).     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.     

Fractionation of stable carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants

The active species of "CO(2)" and the amount of fractionation of stable carbon isotopes have been determined for a partially purified preparation of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) from corn (Zea mays) leaves. The rates of the enzyme reactions, using substrate amounts of HCO(3) (-), CO(2) or CO(2) plus carbonic anhydrase, show that HCO(3) (-) is the active species of "CO(2)" utilized by PEP carboxylase. The K(m) values for CO(2) and HCO(3) (-) are 1.25 mm and 0.11 mm, respectively, which further suggest the preferential utilization of HCO(3) (-) by PEP carboxylase. The amount of fractionation of stable carbon isotopes by PEP carboxylase from an infinite pool of H(12)CO(3) (-) and H(13)CO(3) (-) was -2.03 per thousand. This enzyme fractionation (delta), together with the fractionation associated with absorption of CO(2) into plant cells and the equilibrium fractionation associated with atmospheric CO(2) and dissolved HCO(3) (-) are discussed in relation to the fractionation of stable carbon isotopes of atmospheric CO(2) during photosynthesis in C(4) plants.     

Purification and characterizaton of plastidic pyruvate kinase from developing seeds of Brassica campestris L.

Plastidic pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was purified to near homogeneity as judged by native PAGE with about 4% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive blue Sepharose-CL-6B. The purified enzyme having molecular mass of about 266 kDa was quite stable and showed a broad pH optimum between pH 6.8-7.8. Typical Michaelis-Menten kinetics was obtained for both the substrates with K(m) values of 0.13 and 0.14 mM for PEP and ADP, respectively. The enzyme could also utilize CDP, GDP or UDP as alternative nucleotide to ADP, but with lower Vmax and higher K(m). The enzyme had an absolute requirement for a divalent and a monovalent cation for activity and was inhibited by oxalate, fumarate, citrate, isocitrate and ATP, and activated by AMP, aspartate, 3-PGA, tryptophan and inorganic phosphate. ATP inhibited the enzyme competitively with respect to PEP and non-competitively with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. This inhibition by either ATP or oxalate was not due to chelation of Mg2+, as the inhibition was not relieved on increasing Mg2+ concentration even upto 30 mM. Initial velocity and product inhibition studies demonstrated the reaction mechanism to be compulsory ordered type. The enzyme seems to be regulated synergistically by ATP and citrate.     

Characterization and properties of the pyruvate phosphorylation system of Acetobacter xylinum

The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in Acetobacter xylinum has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg(2+) ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (P(i)) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal pH for PEP synthesis was pH 8.2; for the reversal it was pH 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at pH 8.2 and 0.45 at pH 6.5. The apparent K(m) values of the components of the system in the forward reaction were: pyruvate, 0.2 mm; ATP, 0.4 mm; P(i), 0.8 mm; Mg(2+), 2.2 mm; and for the reverse reaction: PEP, 0.1 mm; AMP, 1.6 mum; PP, 0.067 mm; Mg(2+), 0.87 mm. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (K(i) = 0.2 mm). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by p-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in A. xylinum.     

水稻叶片酸性磷酸酯酶活性及其部分特性

从水稻叶片部分纯化了水解磷酸烯醇式丙酮酸的磷酸酯酶,其Ｋｍ（ＰＥＰ）为０．１ｍｍｏｌ／Ｌ,最适ＰＨ５．３．在偏酸性ＰＨ条件下（ＰＨ４．０～７．２）稳定,对热亦较稳定．酶活性受Ｐｉ强烈抑制．它对其底物要求不专一,能水解多种含磷酯键的化合物．表明它是一种非专一性的酸性磷酸酯酶。各种含磷酯键的代谢物对酶活性起竞争性抑制作用,且表现出叠加性．Ｃｕ（２＋）、Ｚｎ（２＋）和Ｆｅ（２＋）抑制酶活性,Ｍｇ（２＋）、Ｍｎ（２＋）、Ｃａ（２＋）、Ｃｏ（２＋）和ＥＤＴＡ无影响．     

Immunocytochemical study of phosphoenolpyruvate carboxylase in nodulated Alnus glutinosa

The activities of phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.3.1) have been investigated in various organs of young nodulated Alnus glutinosa. The root nodules exhibited the highest specific enzyme activity when compared with the one in roots and leaves. Furthermore, in the root nodules the PEP carboxylase was predominantly localized in the cytosol of the large cortical cells containing the endophyte vesicles.Abbreviations PEP carboxylase phosphoenolpyruvate carboxylase - MDH malate dehydrogenase - PVP polyvinylpyrrolidone - PBS phosphate buffer saline     

Regulation of phosphoenolpyruvate carboxylase from Crassula by interconversion of oligomeric forms

Using size-exclusion high-performance liquid chromatography, it is shown that phosphoenolpyruvate carboxylase from Crassula argentea, a crassulacean acid metabolism (CAM) plant, exists primarily in the form of a tetramer of a 100-kDa subunit at night and as a dimer of the same subunit during the day. The tetrameric enzyme from night leaves is not inhibited by malate, while the dimeric form from day leaves can be completely inhibited by malate. The purified day, or dimer, form of the enzyme can be converted to the tetramer by concentration and exposure to Mg2+. When thus converted, the tetramer is insensitive to malate inhibition, and is more strongly activated by glucose 6-phosphate than the dimer. The purified night, or tetramer, form is converted to the dimer by incubation for 60 min at pH 8.2. This enzyme may also be converted to the dimer by adding 1.5 mM malate to the elution buffer, but preincubation for 15 min with phosphoenolpyruvate prevents disaggregation when chromatographed with buffer containing malate. Preincubation with 1mM EDTA and subsequent chromatography with buffer containing malate shows a progressive dissociation of the tetrameric form with increasing time of preincubation. The implications of these observations for the diurnal regulation of phosphoenolpyruvate carboxylase in CAM metabolism are discussed.     

Purification and Characterization of Phosphoenolpyruvate Carboxylase from Developing Seeds of Brassica

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified to apparent homogeneity with about 29% recovery from developing seeds of Brassica using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sepharose CL-6S. The purified enzyme with mol wt of about 400 kD exhibited maximum activity at pH 8.0. The enzyme had an absolute requirement for a divalent cation which was satisfied by Mg²⁺. The enzyme showed typical hyperbolic kinetics with PEP and HCO^?3 with Km of 0.125 and 0.104 mM, respectively. Glu-6-P could activate the enzyme, whereas other phosphate esters such as fru-1, 6-P₂, L-glycerophosphate and 3-PGA did not have any effect on the enzyme activity. Noneof the amino acids at 5 mM concentration had any significant effect on the enzyme activity. Nucleotide monophosphates and diphosphates did not inhibit the enzyme significantly, whereas ATP inhibited the enzyme activity. Oxaloacetate and malate inhibited the enzyme non-competitively with respect to PEP with Ki values of 0.127 and 1.25 mM, respectively. The enzyme activity in vivo seems to be regulated ’Tlainly by availability of its substrate and activation by glu-6-P, both of which are supplied through glycolysis.     

Regulation of biosynthesis of secondary metabolites

The activity of PEP carboxylase (E.C.4.1.1.31) was demonstrated in cell free extracts ofStreptomyces aureofacines. The enzyme was purified 610 fold. AcetylCoA increased the affinity of the purified enzyme for substrate approximately tenfold and doubled the specific activity of the enzyme preparation. Inorganic phosphate was not essential for the reaction; on the contrary, it had an inhibitory effect. Essential cofactors were divalent cations, the most potent of which was Mn²⁺. The kinetic characters of the purified enzyme were similar to figures for PEP carboxylase from other sources. The substrate saturation function replotted according to the Hill equation showed the extent of intramolecular interactions, reflecting the allosteric nature of the enzyme.     

Junctions of catabolic and anabolic pathways in Zymomonas mobilis: phosphoenolpyruvate carboxylase and malic enzyme

Summary The synthesis of oxalacetate and malate in the ethanol-producing bacterium Zymomonas mobilis have been investigated. Cell-free extracts were examined for pyruvate carboxylase, phosphoenolpyruvate (PEP) carboxylase, PEP carboxytransphosphorylase, PEP carboxykinase, and malic enzyme, but only PEP carboxylase and nicotine adenine dinucleotide (NAD)-dependent malic enzyme activities could be detected. The PEP carboxylase, partially purified from extracts, was not affected by acetyl-coenzyme A. Intermediates of the tricarboxylic acid cycle and aspartate inhibited the enzyme competitively with PEP. Of these, citrate and -ketoglutarate were the strongest inhibitors. The physiological roles of PEP carboxylase and malic enzyme in Z. mobilis are discussed.Dedicated to Prof. Dr. A. Fiechter, ETH Zürich, on the occasion of his 65th birthday     

Fungal metabolism of biphenyl.

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.     

The formation of 5-phosphomevalonate by mevalonate kinase in Hevea brasiliensis latex

1. Evidence has been produced for the formation of 5-phosphomevalonate from potassium dl-mevalonate by the latex of Hevea brasiliensis and by reconstituted freeze-dried serum obtained from this latex. 2. The enzyme, mevalonate kinase, catalysing the formation of 5-phosphomevalonate from potassium dl-mevalonate and ATP has been partially purified. 3. 5-Phosphomevalonate formed by the purified mevalonate kinase from potassium [2-(14)C]mevalonate has been shown to be incorporated by latex into rubber to about 2.4 times the extent of dl-mevalonate. 4. The enzyme can utilize inosine triphosphate as effectively as adenosine triphosphate as a phosphate donor and is also slightly active with uridine triphosphate. 5. The enzyme was fairly stable to a range of pH values and temperatures, the activity being optimum at pH7.5 and 60-70 degrees . The energy of activation was 10.7kcal./mole. The K(m) values were 0.13mm for potassium dl-mevalonate and 2.0mm for ATP at 30 degrees . 6. The enzyme required the presence of Mn(2+) (1mm) for maximum activity; this could be replaced by Mg(2+) (4mm), which was less effective, and by Ca(2+), which was far less effective. 6. Although the enzyme did not require cysteine or reduced glutathione for activation in aerobic conditions, it was inhibited by reagents known to react with thiol groups.     

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.     

Coproporphyrinogenase in tobacco (Nicotiana tabacum L.)

1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent K(m) value of 3.6x10(-5)m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30mug/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe(2+) concentrations up to 0.5mm, beyond which inhibition occurred. Co(2+) and Mn(2+) were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe(3+) and Cu(2+), both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Partial Characterization of Cell-bound Lipase of Candida paralipolytica

In accordance with the regulation by aspartate of phosphoenolpyrubate (PEP*) carboxylase, glutamate formation in Brevibacterium flavum, a glutamate-producing bacterium, was inhibited by the addition of aspartate. Furthermore, an increase in aspartate formation caused by a mutational decrease in citrate synthase specific activity was accompanied by a decrease in the total amount of glutamate and aspartate formed. However, a mutational decrease in glutamate dehydrogenase activity caused a decrease in the total amount without increasing the asparate formation but with accumulation of 2-oxoglutarate, suggesting that the feedback inhibition by the aspartate of PEP carboxylase was enhanced by 2-oxoglutarate. In fact, partially purified PEP carboxylase from this organism was found to be synergistically inhibited by aspartate and 2-oxoglutarate, citrate, cis-aconitase, or isocitrate. Among them, the effects of tricarboxylic acids were attributed to their non-specific chelating action with Mn²⁺, an activator of the enzyme. The synergistic action of 2-oxoglutarate was accompanied by a decrease in Hill coefficient for the aspartate of the enzyme.