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Partial Purification and Properties of L-Glutamine D-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus¹

^a Department of Biochemistry, University of California, Berkeley, California 94720

L-Glutamine D-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for L-glutamine as an amide nitrogen donor, and L-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mM and 0.5 mM were obtained for D-fructose 6-phosphate and L-glutamine, respectively. The enzyme was competitively inhibited with respect to D-fructose 6-phosphate by uridine diphosphate-N-acetyl-D-glucosamine which had a Ki value of 13 µM. Upon removal of L-glutamine and its replacement by D-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-D-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-D-glucosamine acts as a feedback inhibitor of the enzyme.

¹ This investigation was supported in part by Research Grant A-1418 from the National Institutes of Health, United States Public Health Service, and by Research Grant GB 11819 from the National Science Foundation. Support of this work by the Agricultural Experiment Station is also acknowledged.

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