Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima

Abstract

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 (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium 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 microM, respectively; the apparent Vmax values (at 55 degrees 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 degrees C and was not inactivated by heat upon incubation at 80 degrees 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 (alpha2) 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 Vmax values (at 50 degrees 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 degrees 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 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

FIG. 1

Analysis of PTA from T. maritima by SDS-PAGE at various steps of the purification procedure. Fractions with the highest specific activities obtained after various chromatographic steps (see Materials and Methods) were used. Protein was denatured in SDS and separated in 14% polyacrylamide slab gels (8 by 7 cm) (19), which were stained with Coomassie brilliant blue R 250. Lanes: 1 and 8, molecular mass standards (Sigma); 2, 100,000 × g supernatant, 12 μg of protein; 3, DEAE-Sepharose, 10 μg of protein; 4, Q-Sepharose, 10 μg of protein; 5, phenyl-Sepharose, 8 μg of protein; 6, Superdex 200, 6 μg of protein; 7 Mono Q, 2 μg of protein. The positions of molecular mass standards are indicated on the left.

FIG. 2

Effect of temperature on the specific activity of PTA from T. maritima. (A) Temperature dependence of the specific activity. (B) Arrhenius plot of the same data. Enzyme activity was measured in the direction of acetyl phosphate formation from acetyl-CoA (see Materials and Methods). The assay mixture contained 0.087 μg of protein. T, temperature.

FIG. 3

Analysis of AK from T. maritima by SDS-PAGE at various steps of the purification procedure. Fractions with the highest specific activities obtained after various chromatographic steps (see Materials and Methods) were used. Protein was denatured in SDS and separated in 14% polyacrylamide slab gels (8 by 7 cm) (19), which were stained with Coomassie brilliant blue R 250. Lanes: 1 and 8, molecular mass standards (Sigma); 2, 100,000 × g supernatant, 12 μg of protein; 3, DEAE-Sepharose, 10 μg of protein; 4, Q-Sepharose, 7 μg of protein; 5, phenyl-Sepharose, 5 μg of protein; 6, Superdex 200, 3 μg of protein; 7, Mono Q, 2 μg of protein. The positions of molecular mass standards are indicated on the left.

FIG. 4

Effect of various salts on the thermostability of AK from T. maritima at 100°C. The 0.1-ml incubation mixtures contained 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.36 μg of enzyme. Salts (1 M) were included as indicated. At the times indicated, the remaining enzyme activity was measured at 55°C in the direction of acetyl phosphate formation (pyruvate kinase/lactate dehydrogenase assay).