Purification and properties of acetyl-CoA synthetase (ADP-forming), an archaeal enzyme of acetate formation and ATP synthesis, from the hyperthermophile Pyrococcus furiosus.

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 Pi (acetyl-CoA + ADP + Pi --> acetate + ATP + CoA) [Schifer, 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 alpha2beta2 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 Pi to acetate, ATP and CoA. The apparent Vmax value in the direction of acetate formation was 18 U/mg (55 degrees C), the apparent Km values for acetyl-CoA, ADP and Pi were 17 microM, 60 microM and 200 microM, respectively. ADP and Pi could not be replaced by AMP and PPi, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent Vmax value in the direction of acetyl-CoA formation was about 40 U/mg (55 degrees C), and the apparent Km values for acetate, ATP and CoA were 660 microM, 80 microM and 30 microM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (100%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (100%), 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 (100%), GDP (220%) and IDP (250%) were used, and in addition to ATP (100%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg2+, which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90 degrees 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 degrees C.