Acetyl-CoA synthetase (ADP-forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and P, (acetyl-CoA + ADP + P, -acetate + ATP + CoA) [Schafer, T., Selig, M. & Schonheit, P. (1993) Arch. Microbiol. 159, 72-83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96-fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen-stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an a2,B2 structure. The N-terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl-CoA, ADP and P, to acetate, ATP and CoA. The apparent V,,, value in the direction of acetate formation was 18 U/mg (55 "C), the apparent K,,, values for acetylCoA, ADP and P, were 17 pM, 60 pM and 200 FM, respectively. ADP and P, could not be replaced by AMP and PP,, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent V,,, value in the direction of acetyl-CoA formation was about 40 U/mg (55"C), and the apparent K, values for acetate, ATP and CoA were 660 pM, 80 pM and 30 pM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (loo%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (loo%), butyrate (92%), isobutyrate (79 %), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl-CoA synthetase (ADP-forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (< 3 %). In addition to ADP (loo%), GDP (220%) and IDP (250%) were used, and in addition to ATP (loo%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg", which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90"C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KCI (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100°C.Keywords: acetyl-CoA synthetase (ADP-forming) ; acetate formation; Archaea; Pyrococcus furiosus; hyperthermophiles.Acetyl-CoA synthetase (ADP-forming) catalyzes the following reversible reaction: acetyl-CoA + ADP + Pi * acetate + ATP + CoA. This unusual synthetase was first detected in the eukaryotic parasite Entamoeba histolytica and later in the anaerobic protozoon Giardia lamblia, where it is involved in acetate formation and ATP production during fermentative metabolism (Reeves et al., 1977;Lindmark, 1980; reviewed by Muller, 1988;Adam, 1991).In prok...

Methanosarcina barkeri (strain Fusaro) was grown on pyruvate as methanogenic substrate [Bock, A. K., Prieger-Kraft, A. & Schonheit, P. (1994) Arch. Microbiol. 161, 33-46]. The first enzyme of pyruvate catabolism, pyruvate oxidoreductase, which catalyzes oxidation of pyruvate to acetyl-CoA was purified about 90-fold to apparent electrophoretic homogeneity. The purified enzyme catalyzed the CoA-dependent oxidation of pyruvate with ferredoxin as an electron acceptor which defines the enzyme as a pyruvate:ferredoxin oxidoreductase. The deazaflavin, coenzyme F,,,,, which has been proposed to be the physiological electron acceptor of pyruvate oxidoreductase in methanogens, was not reduced by the purified enzyme. In addition to ferredoxin and viologen dyes, flavin nucleotides served as electron acceptors. Pyruvate : ferredoxin oxidoreductase also catalyzed the oxidation of 2-oxobutyrate but not the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, 3-hydroxypyruvate and oxaloacetate. The apparent K,,, values of pyruvate:ferredoxin oxidoreductase were 70 pM for pyruvate, 6 pM for CoA and 30 pM for clostridial ferredoxin. The apparent V,,,, with ferredoxin was about 30 U/mg (at 37°C) with a pH optimum of approximately 7. The temperature optimum was approximately 60°C and the Arrhenius activation energy was 40 kJ/mol (between 30°C and 60°C). The enzyme was extremely oxygen sensitive, losing 90% of its activity upon exposure to air for 1 h at 0°C. Sodium nitrite inhibited the enzyme with a K, of about 10 mM. The native enzyme had an apparent molecular mass of approximately 130 kDa and was composed of four different subunits with apparent molecular masses of 48, 30, 25, and 15 kDa which indicates that the enzyme has an a y 6 structure. The enzyme contained 1 mol/mol thiamine diphosphate, and about 12 mol/mol each of non-heme iron and acid-labile sulfur. FAD, FMN and lipoic acid were not found. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the a-subunit was similar to the N-terminal amino acid sequence of the a-subunit of the heterotetrameric pyruvate : ferredoxin oxidoreductases of the hyperthermophiles Archaeoglobus fulgidus, Pyrococcus furiosus and Thermotoga maritima and of the mesophile Helicobacter pylori, and to the N-terminal amino acid sequence of the homodimeric pyruvate: ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the a-subunit of the M. barkeri enzyme and the heterodimeric pyruvate: ferredoxin oxidoreductase of the archaeon Halobacter~~m halobiurn.

Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (α4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases fromClostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi ⇌ acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 μM, respectively; the apparentV max values (at 55°C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90°C and was not inactivated by heat upon incubation at 80°C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (α2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP ⇌ acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent V max values (at 50°C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90°C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4(1 M), which was most effective, the enzyme did not lose activity upon incubation at 100°C for 3 h. The temperature optimum at 90°C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

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