Asparagine Biosynthesis in Soybean Nodules

Publisher Website
Google Scholar
Abstract
Asparagine biosynthesis in soybean (G. max [L.] Merr.) nodules has been difficult to demonstrate due to the poor conversion of suspected immediate precursors to asparagine and the instability of the key enzyme asparagine synthetase. The effects of 2 ammonium assimilation inhibitors on the metabolism of 14CO2 to [14C]asparagine and the existence in nodules of the enzyme asparagine synthetase were studied. When detached nodules were incubated in 14CO2, radioactivity in asparagine (as a percentage of amino acid cpm) increased 10-fold over 4 h. Vacuum infiltration of 10 mM methionine sulfoximine or 10 mM azaserine prior to 14CO2 incubations decreased both the rate of dark fixation and the radioactivity in the amino acid fraction. These inhibitors also decreased the recovery of label in aspartate and asparagine. These results, plus the sequence of labeling of metabolites from 14CO2, are consistent with a glutamine-dependent synthesis of asparagine from aspartate with oxalacetate as a precursor to aspartate. An enzyme catalyzing the ATP- and glutamine-dependent amidation of aspartic acid to form asparagine was isolated from soybean nodules. High levels of SH protectants were required and the inclusion of glycerol and substrates in the extraction buffer helped to stabilize the enzyme. Enzyme activity in taproot nodules increased between 38 and 41 days after planting and peaked soon after flower initiation (45 days). The activity then declined to basal levels by 70 days. On a total enzyme activity basis, there was 170-fold more asparagine synthetase activity in the infected zone of the nodule than in the cortex and 205-fold more activity in the cytosol than the bacteroid fraction. The enzyme has a broad pH maximum around pH 8.25, and the apparent Km values for the substrates aspartate, MgATP, and glutamine are 1.24, 0.076 and 0.16 mM, respectively. Ammonium ion can replace glutamine as the N donor, but the Km value of the enzyme for ammonium ion is 40-fold higher than that for glutamine.