Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster: Purification and properties

Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster has been purified 1400-fold over a crude 230,000g supernatant fraction. The optimum activity of the enzyme occurs at magnesium concentrations above 10 mm at 37 °C and pH 7.5. At a 50 mm Mg²⁺ concentration, NH₄⁺ stimulates the ATP-PP₁ exchange reaction as much as 2-fold. Ammonium chloride causes an increase in the V with no change in the K_m with phenylalanine as substrate. Homologous (Drosophila) tRNA, in the presence of NH₄⁺, further stimulates the ATP-PP_i, exchange reaction but inhibits the reaction in the absence of NH₄⁺.In the presence of its substrates the enzyme is inactivated by NEM to varying degrees depending upon the substrate or combinations of substrates used. In the presence of phenylalanine the enzyme is partially protected but both ATP and tRNA make the enzyme more susceptible to inactivation. NEM together with ATP and tRNA or all three substrates results in near-total inactivation.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Altered Kinetic Properties of Tyrosine-183 to Cysteine Mutation in Glutamine Synthetase of <Emphasis Type="Italic">Anabaena variabilis</Emphasis> Strain SA1 Is Responsible for Excretion of Ammonium Ion Produced by Nitrogenase

A L-methionine-D,L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N₂ reduction. In order to determine the biochemical basis for the NH₄ ⁺-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn⁺² for optimum activity, while SA0-GS was Mg⁺² dependent. SA0-GS had the following apparent K _m values at pH 7.5: glutamate, 1.7 mM; NH₄ ⁺, 0.015 mM; ATP, 0.13 mM. The apparent K _m for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 mM; NH₄ ⁺, 12.4 mM; ATP, 0.17 mM). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH₄ ⁺-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K _m values for NH₄ ⁺ and glutamate, and thus limited the assimilation of NH₄ ⁺ generated by N₂ reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH₄ ⁺ binding and metal ion coordination. Received: 3 July 2002 / Accepted: 29 July 2002     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Expression Optimization and Biochemical Characterization of a Recombinant γ-Glutamyltranspeptidase from Escherichia coli Novablue

A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His₆-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K _m and V _max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10⁻³ mM min⁻¹, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM L-Gln, 40 mM ethylamine, and 1.04 U His₆-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Continuous production of L-aspartic acid by immobilized aspartase

Various methods were tried for the immobilization of aspartase, and the preparation having the highest activity was obtained when partially purified aspartase from Escherichia coli was entrapped into polyacrylamide gel Iattice. Enzymatic properties of the immobilized aspartase were investigated and compared with those of the native aspartase. With regard to optimum pH, temperature, concentration of Mn⁺⁺, kinetic constants and heat stability, no marked difference was observed between the native and immobilized aspartases. By employing an enzyme column packed with the immobilized aspartase, conditions for continuous production of L -aspartic acid from ammonium fumarate were investigated. When a solution of 1M ammonium fumarate (pH 8.5, containing 1mM MnCl₂) was passed through the aspartase column at the flow rate of SV = 0.08 at 37°C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in a good yield.     

Mannitol Biosynthesis in Pyrenochaeta terrestris II. D-Mannitol-l-phosphatase

Cell-free extracts of mycelial mats of Pyrenochaeta terrestris contained an enzyme which hydrolyzed mannitol-l-phosphate to mannitol and inorganic phosphate. Greatest mannitol-1-phosphatase activity occurred early in the growth period when the mannitol content of the mats was at a maximum. The enzyme was active over a broad pH range with optimum activity between pH 6.5–7.0 in 0.05 M Tris-maleate buffer. Maiinitnl-1-phosphatase was inhibited by reagents known to inhibit enzymes containing -SH groups. A 10-fold purification was attained by a combination of (NII₄)₂ SO₄ fractionation and gel filtration on Sephadex G-100. The partially purified enzyme required Mg^?2 for activity and did not hydrolyze a number of sugar phosphates. Km values for mannitol-l-phosphate and Mg^?2 with the partially purified extract were 3 × 10^?3 M and 1 × 10^?4 M respectively.     

Purification and Properties of Gitrus natsudaidai Pectinesterase

The level of glutamine synthetase in Micrococcus glutamicus ATCC 13032 varied in response to the nitrogen source in culture medium; it was 10?20 fold higher in glutamate-, peptone- or yeast extract-grown cells than in ammonia- or urea-grown cells. Ammonia (3 mM) reduced the enzyme level to 50% when added to glutamate medium. No difference between nitrogen sources was observed in extent of inhibition by Mg²⁺ of γ-glutamylhydroxamate-forming (transferring) reaction in crude extracts.

The optimum pH was 7.0 ? 8.0 for glutamine-forming (synthesizing) reaction and 7.0 for transferring reaction. The enzyme was stable to heating at 50°C for 10 min in 0.05 M potassium phosphate buffer (pH 6.0) containing 0.1 mM MnCl₂. Km values for glutamate, ammonia and ATP in synthesizing reaction were 7.9, 5.0 and 1.2 mM, respectively. GTP and hydroxylamine could be substituted for ATP and ammonia with about 10 and 30% reactivity. Mg²⁺ was effective as a cofactor in synthesizing reaction and Mn²⁺ showed 34% of the reactivity of Mg²⁺ at a concentration of 30 mM. Glutamine synthetase was inhibited by adenosine, AMP and ADP but not by amino acids other than D-threonine. The regulation system of glutamine synthetase in M. glutamicus is discussed.     

Purification and characterization of chorismate mutase isoenzymes from ruta graveolens l.

Two isoenzymes of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, were isolated and partially purified from suspension-cultured cells of Ruta gravelens by DEAE-sephacel chromatography and gel filtration. 60–72% of the total activity measured after DEAE-sephacel chromatography were obtained as CM-1 and 28–40% were CM-2 activity. CM-1 was inhibited by phenylalanine (K₁ = 4 · 10^?6 M) and tyrosine (K₁ = 8. 10^?6M) and activated by tryptophan. In contrast, CM-2 was not influenced by these three amino acids. The molecular weights estimated by gel filtration on SEPHADEX G-150 were 56000 for CM-1 and 45000 for CM-2, respectively. Both isoenzymes were stable at ?20°C, but exhibited different behaviour during thermal inactivation and different optima of reaction temperature. CM-1 catalysed the reaction at a pH optimum of pH 7.8 and CM-2 showed a broad optimum between 6–10. The K_m-values for chorismic acid were determined to be 1.1 mM for CM-1 and 0.5 mM for CM-2. The isoenzymes showed different behaviour to inhibitors of sulfhydryl groups. There were no differences in all parameters of chorismate mutase examined for two various cell lines of Ruta graveolens.     

Studies on γ-Glutamyl Transpeptidase in Green Asparagus (Asparagus officinalis L.)

Presence of γ-glutamyl transpeptidase in soluble cell fraction of green asparagus has been confirmed for the first time. The partially purified enzyme, having an optimal pH ca. 8.0 for the enzymatic reaction, was labile for storage at alkaline pH. K_m for the substrate γ-glutamyl aniline was 1.1 × 10^?3m. Although tris-acetate—sodium citrate buffer stimulated the activity, tris-HCl, veronal, or phosphate was poor buffers and an addition of Mg⁺⁺ was not so effective. Among amino compounds tested as acceptors, methionine and phenylalanine were effective, leucine and basic amino acids poorly effective, and glycylglycine non-effective, whereas glutathione and acidic amino acids were inhibitory.     

THE RELATION BETWEEN BIOCHEMICAL AND MORPHOLOGICAL DIFFERENTIATION IN BLASTOCLADIELLA EMERSONII. I. ENZYMATIC SYNTHESIS OF GLUCOSAMINE-6-PHOSPHATE

Lovett , James S., and Edward C. Cantino . (Michigan State U., East Lansing.) The relation between biochemical and morphological differentiation in Blastocladiella emersonii. I. Enzymatic synthesis of glucosamine-6-phosphate. Amer. Jour. Bot. 47(6): 499–505. Illus. 1960.—The enzyme glucosamine synthetase (glutamine-fructose-6-phosphate transamidase) was purified ca. 19-fold from extracts of the aquatic phycomycete, Blastocladiella emersonii, by centrifugation, protamine sulfate fractionation, and adsorption on Ca₃(PO₄)₂ gel The pH optimum, time course, and relation between enzyme concentration and reaction rate were established for the partially purified synthetase. The reaction was the same as that of the enzyme of Neurospora crassa: D-fructose-6- phosphate + L-glutamine —> D-glucosamine-6-phosphate + L-glutamic acid. The 20-fold purification attained resulted in an enzyme preparation with a specific activity 40 times greater than that reported for other organisms to date.     

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l -glutamate: ammonia ligase, ADP-forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn²⁺- and Mg²⁺-dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg²⁺ and β-mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg²⁺ and ATP. One-tenth mM phenylmercuric acetate inhibited the Mg²⁺-dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The K_m values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH₂OH, respectively. The constant for NH⁺₄ was an order of magnitude higher in the presence of Mn²⁺ than Mg²⁺. When the amino acids were externally added to the reaction mixtures, the measurement of P_i exhibited a higher degree of enzyme inhibition than the measurement of γ-glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg²⁺- and Mn²⁺-dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l -form) inhibited the enzyme 30% in the presence of either Mg²⁺ or Mn²⁺. Aspartate (Mg²⁺-dependent) and histidine (Mn²⁺-dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg²⁺-dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.     

Metabolism of Aromatic Amino Acid in Microorganisms

Phenylalanine ammonia-lyase, which catalyzes the conversion of l-phenylalanine to trans-cinnamic acid and ammonia, has been partially purified from the cells of Rhodotorula. Some of the properties of this phenylalanine ammoyia-lyase were investigated. The enzyme was stable in phosphate buffer of pH over the range of 6.0 to 7.0 On heating, the enzyme was stable up to 50°C, but above 60°C, it was destroyed. The enzyme activity was strongly inhibited by p-chloromercuribenzoate at 10^?5 m and almost recovered by the addition of glutathione or mercaptoethanol at 10^?3 m. The present enzyme preparation of Rhodotorula also catalyzed the deamination of l-tyrosine to trans-p-coumaric acid. trans-p-Coumaric acid was isolated from the reaction mixture and identified by its absorption spectra. The rates of deamination showed optima at pH 9.0 and 9.5 for l-phenylalanine and l-tyrosine, respectively.     

Mutation affecting the charging reaction of alanyl-tRNA synthetase from Escherichia coli K 10

Summary A temperature-sensitive mutant of Escherichia coli was identified as having an altered alanyl-tRNA synthetase. Specific activity of wild type and mutant cell-free extracts showed no difference in the hydroxamate assay; the charging activity, however, was more than 10 fold lower for mutant extract protein. Wild type alanyl-tRNA synthetase has been purified 344 fold, the mutant enzyme was enriched 45 fold. With these preparations the following results were obtained:Sedimentation analysis in sucrose gradients indicates a molecular weight of the mutant enzyme of half the size of the wild type enzyme. Analytical gel filtration yields an approximate size for the native enzyme of 165000 and for the mutant enzyme material of 95,000. The mutant alanyl-tRNA synthetase differs from the wild type enzyme by a 10 fold increase in the k _mfor tRNA; no true difference in the k _m-values for the other substrates was detected. Temperature studies indicate an unusual low temperature-optimum for the charging reaction of both enzymes, whereas hydroxamate fromation capacity increases linearly up to almost 50°C. High temperature treatment of the native enzyme selectively affects the aminoacylation reaction but not the activation step; no effect of such treatment of the mutant enzyme was detected. It is proposed that the mutation causes the enzyme to dissociate and that the resulting subunits possess and altered tRNA binding site.     

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M_r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the K_m ATP is 1.1 mm but the K_m for l-glutamate is not altered. The effect of pH on the K_m for ammonium indicates that NH₃ rather than NH₄⁺ may be the true substrate. At 10 mm Mg²⁺, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg²⁺ concentrations produce progressively more acidic optima while lower Mg²⁺ concentrations raise the pH optimum. The rate-response curve for Mg²⁺ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (K_i, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K_i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.     

Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10^?4, 226.7 ×10^?4 and 33.8 × 10^?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent K_m values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.     

Purification and characterization of benzoyl-CoA ligase from a syntrophic,benzoate-degrading,anaerobic mixed culture

Summary The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl₂; MgCl₂ in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K _m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V _max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K _m of 0.17 mM and a V _max of 1.05 U/mg and for ATP a K _m of 0.16 mM and a V _max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K _i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found. Correspondence to: J. Winter