Acyl coenzyme A synthetase from Pseudomonas fragi catalyzes the synthesis of adenosine 5'-polyphosphates and dinucleoside polyphosphates

Abstract

Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5'-tetraphosphate (p4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5'-O-[gamma-thiotriphosphate] (ATP gamma S), adenosine(5')tetraphospho(5')adenosine (Ap4A), and adenosine(5')pentaphospho(5')adenosine (Ap5A) are also substrates of the reaction yielding p4(d)A in the presence of tripolyphosphate (P3). UTP, CTP, and AMP are not substrates of the reaction. The K(m) values for ATP and P3 are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl2 or CoCl2 equimolecular with the sum of ATP and P3. The relative rates of synthesis of p4A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p4A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p4A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap4A (from ATP), Ap5A (from p4A), and adenosine(5')tetraphospho(5')nucleoside (Ap4N) from adequate adenylyl donors (ATP, ATP gamma S, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates).

FIG. 1

(A) Synthesis of p₄A and p₅A from ATP, P₃, and P₄ catalyzed by acyl-CoA synthetase. The reaction mixtures contained 1.15 mM ATP, 0.8 μl of desalted inorganic pyrophosphatase, 5 mM P₃ (lanes 2 to 6) or P₄ (lanes 7 to 11), and acyl-CoA synthetase (4.9 μg of protein when the polyphosphate added was P₃ or 9.8 μg of protein when it was P₄); other conditions and TLC analysis procedures were as described in Materials and Methods. Lanes: 1 and 12, standards of p₄A, ATP, ADP, AMP, and adenosine; lanes 2 and 7, the control mixtures without acyl-CoA synthetase after 2 and 4 h of incubation, respectively; lanes 3 to 6, the complete mixture containing P₃ taken after 0, 0.5, 1, and 2 h of incubation, respectively; lanes 8 to 11, the complete mixture containing P₄ taken after 0, 1, 2, and 4 h of incubation, respectively. (B and C) Effect of alkaline phosphatase on the presumptive p₄A (B) and p₅A (C) synthesized. Similar reaction mixtures (0.8 ml) were incubated for 7 or 36 h (in the case of P₃ or P₄ as adenylyl acceptor substrate, respectively), and the presumptive p₄A or p₅A formed was purified (see Materials and Methods) and characterized as follows: reaction mixtures (1 ml) containing 50 mM MES-KOH (pH 6.7), 0.2 mM MgCl₂, and purified p₄A (100 μM) or p₅A (60 μM) were treated with alkaline phosphatase (0.5 μg of protein); at the times indicated, aliquots were taken and analyzed by HPLC. The numbers 0 to 5 on the top of the chromatographic peaks correspond to adenosine, AMP, ADP, ATP, p₄A, and p₅A, respectively.

FIG. 2

Coelution of p₄A and palmitoyl-CoA synthetic activities upon gel filtration. A sample of acyl-CoA synthetase was applied to a Bio-Sil-Sec 250 column as described in Materials and Methods (inset); the arrow marks the column void volume; peaks p and a correspond to protein and adenine nucleotides, respectively. The activities of synthesis of p₄A (•) and palmitoyl-CoA (○) were studied with 15 and 0.33 μl of the column fractions, respectively; [2,8-³H]ATP was used as radioactive substrate. Other conditions were as described in Materials and Methods. The broken line represents absorbance at 280 nm.

FIG. 3

Effect of pH on the synthesis of p₄A and palmitoyl-CoA catalyzed by acyl-CoA synthetase. The reaction mixtures (50 μl) contained 50 mM MES-KOH (pH 5.5 and 6.3), HEPES-KOH (pH 7.2), or Tris-HCl (pH 8.2) and [2,8-³H]ATP as radioactive substrate. In the case of p₄A synthesis, 8.3 μg of protein was used; in the case of palmitoyl-CoA synthesis, the enzyme amount varied between 2.0 (pH 5.5) and 0.4 (pH 8.2) μg of protein; other conditions were as described in Materials and Methods.

FIG. 4

Effect of asymmetrical dinucleoside tetraphosphatase on Ap₄A (left panel) or Ap₅A (right panel) obtained from ATP or p₄A, catalyzed by acyl-CoA synthetase. Reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, and purified (see Materials and Methods) Ap₄A or Ap₅A (33 μM) were treated with asymmetrical dinucleoside tetraphosphatase (0.4 or 0.7 mU/ml, respectively). At the times indicated, aliquots were taken and analyzed by HPLC.

FIG. 5

Spectra of dinucleoside polyphosphates synthesized by acyl-CoA synthetase. Ap₄G and Ap₄C were synthesized as described in Materials and Methods; Ap₄dT and Ap₄X were synthesized as described for Fig. 6. These spectra were obtained with HPLC ChemStation (Hewlett-Packard) from the files produced by the same program during the analysis of the reaction mixtures by HPLC.

FIG. 6

Synthesis of Ap₄N with octanoyl-AMP (left panels) or ATPγS (right panels) as adenylyl donor. The reaction mixtures (90 μl) contained 50 mM MES-KOH (pH 5.5), 0.1 mM dithiothreitol, 6 mM MgCl₂, 1 mM octanoyl-AMP (peak 1′) or ATPγS (peak 3′), 5 mM NTP, and dialyzed acyl-CoA synthetase (26 μg of protein). At the times indicated, aliquots were withdrawn and analyzed by HPLC.

FIG. 7

Effect of CoA and organic acids on the synthesis of p₄A catalyzed by acyl-CoA synthetase. (A) Effect of CoA on the synthesis of p₄A. The reaction mixture (50 μl) contained 50 mM MES-KOH (pH 6.3), 0.1 mM dithiothreitol, 11 mM MgCl₂, 1 mM [2,8-³H]ATP, 10 mM P₃, and the indicated concentrations of CoA and acyl-CoA synthetase (8.3 μg of protein). (B) Effect of organic acids on the inhibitory effect of CoA on the synthesis of p₄A. Reaction mixtures (28 μl) containing 72 mM MES-KOH (pH 6.3), 0.14 mM dithiothreitol, 7.9 mM MgCl₂, 0.76 mM [α-³²P]ATP, 7.2 mM P₃, 0.14 mM CoA, 0.7 μl of desalted inorganic pyrophosphatase, and acyl-CoA synthetase (5.2 μg of protein) were preincubated at 30°C for 20 min. Thereafter, they were supplemented with 12 μl of the following solutions: water (lane b), 1% Triton X-100–5% ethanol (solution C; lane c), 1 mM solutions of palmitic acid in solution C (lane d), octanoic acid in water (lane e), or other possible effectors (acetic acid, lysine, methionine, phenylalanine, tryptophan, and luciferin; lanes f to k, respectively). One hour after the addition of organic acids, aliquots of the reaction were analyzed by TLC. Control without acyl-CoA synthetase is shown in lane a.