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Partial Purification and Properties of D-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus¹

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

D-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for D-glucosamine-6-P. Neither D-glucosamine nor D-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 x 10^–2M EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 x 10^–4M and 7.1 x 10^–4M were obtained for D-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 x 10^–4Mp-hydroxymercuribenzoate, and this inhibition was partially reversed by L-cysteine; indicating the presence of sulfhydryl groups at or near the active site 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 GB11819 from the National Science Foundation. Support of this work by the Agricultural Experiment Station is also acknowledged.