Purification and characterization of glutamine:fructose 6-phosphate amidotransferase from rat liver.
The enzyme glutamine:fructose 6-phosphate amidotransferase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The K(M) values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4, 4'-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the K(inact) of 5.0 microM. Kinetic studies also indicated that 4,4'-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801-810).
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Searle Discovery Research, The Monsanto Life Sciences Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, USA. quang.k.huynh@monsanto.com
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004220:Disulfides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D004220:Disulfides; D010446:Peptide Fragments; D011725:Pyridines; C007542:4,4'-dipyridyl disulfide; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D004229:Dithiothreitol