PURIFICATION AND CHARACTERIZATION OF GLUTAMINE-SYNTHETASE FROM THE COMMERCIAL MUSHROOM AGARICUS-BISPORUS

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30 degrees C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20 degrees C. The enzyme exhibits a K-m for glutamate, ammonium, and ATP of 22 mM, 0.16 mM and 1.25 mM respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10 mM concentrations of L-alanine, L-histidine, L-tryptophan, anthranilic acid, and 5'-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K-m-values were 890 mu M and 240 mu M for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K-i was 17 mu M for both methionine sulfoximine and phosphinothricine.