Purification and Properties of Acetyl‐CoA Synthetase (ADP‐forming), an Archaeal Enzyme of Acetate Formation and ATP Synthesis, from the Hyperthermophile Pyrococcus furiosus

Abstract

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...

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

“…Whatever the role of the N-terminal domain may be, it is likely to also play a role in sensing acetyl-CoA. This conclusion was drawn on the basis of results from isothermal calorimetry experiments, which showed that SePat binds two molecules of acetyl-CoA: one binds to the N-terminal domain, and the other one binds to the catalytic domain (70,(72)(73)(74)(75)(76)(77).…”

“…ADP-forming acyl-CoA synthetases have been characterized in archaea and protists (75)(76)(77). In spite of this homology, no catalytic activity has been attributed to the N-terminal domain of type I or type II GNATs (73,74).…”

Genomic reconstruction of a novel, deeply branched sediment archaeal phylum with pathways for acetogenesis and sulfur reduction

“…The genomes all possessed the ADP-forming subunit of acetyl-CoA synthetase that has been seen to catalyze acetate production in Pyrococcus furiosus and other archaea (Glasemacher et al, 1997). The presence of this enzyme suggests that acetyl-CoA could be converted to acetate; however genes for ethanol fermentation did not appear to be present.…”

Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer

“…ACD and SCS enzymes are able to bind and convert all purine nucleotides at site II, but some enzymes exhibit specificity for either the Ado or Guo nucleotide (2,21,24,38,40). Kinetic characterization of ckcACD1 revealed that both nucleotides are accepted as substrates (SI Appendix, Fig.…”

“…ACDs have been studied in detail in hyperthermophilic archaea, where they function as the major energy-conserving enzymes in the course of anaerobic sugar and peptide fermentation (1)(2)(3)(4). It is believed that ACDs represent a primordial mechanism of ATP synthesis in the early evolution of life.…”