Physical and chemical characterization of glutamine synthetase purified from Mycobacterium phlei.
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 smegmatis 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 degrees C for 2 h or 37 degrees C for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37 degrees 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 degrees 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 (E10.7) was remarkably lower than that of the slightly adenylylated enzyme (E2.5); 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.
Kimura
K
K
Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University, Tokyo.
Suzuki
H
H
Nakano
Y
Y
D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D005974:Glutamate-Ammonia Ligase / Q000032:analysis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009161:Mycobacterium / Q000201:enzymology*; D009168:Mycobacterium phlei / Q000201:enzymology*; D013696:Temperature
D000596:Amino Acids; D001426:Bacterial Proteins; D005974:Glutamate-Ammonia Ligase; D000225:Adenine; D000645:Ammonium Sulfate