Physical and Chemical Characterization of Glutamine Synthetase Purified from Mycobacterium phlei

抄録

Glutamine synthetase (L-glutamate: ammonia ligase [ADP forming]) [EC 6. 3. 1. 2] has been purified from a Gram-positive, acid-fast bacterium, Mycobacterium phlei, by simple procedures with 57% recovery. The enzyme resembled that from Mycobacterium smeg-matis in the subunit size (56, 000), molecular weight (670, 000), amino acid composition, the amino acid sequence of the NH₂-terminal, and the secondary structure. The enzyme activity was regulated by adenylylation of each subunit in the dodecameric molecule. M. phlei glutamine synthetase possesses two useful characteristics: high thermostability and resistance to protease digestion. The enzyme was not inactivated on exposure to 60°C for 2 h or 37° for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37°C for 12 h, pH 7.8. With saturating substrate levels, the Arrhenius plot was nonlinear and concave downward with an intersection point at 45°C, and the activation energies were calculated to be 3.2 and 9.6 cal/mol from the slopes. The specific activity of the highly adenylylated enzyme (E_10.7) was remarkably lower than that of the slightly adenylylated enzyme (E^2.5); however, both enzymes show similar profiles of the Arrhenius plot. These results indicate that the adenylylation of the enzyme does not affect its activation energies.