Purification and characterization of glutamine:fructose 6-phosphate amidotransferase from rat liver
| Autor: | Q. Khai Huynh, Eric A. Gulve, Titik Dian |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Pyridines
Biophysics Fructose 6-phosphate Biology Biochemistry Rats Sprague-Dawley chemistry.chemical_compound Structure-Activity Relationship Glucosamine Sequence Analysis Protein Animals Humans Enzyme kinetics Amino Acid Sequence Disulfides Molecular Biology Polyacrylamide gel electrophoresis Glutamine amidotransferase chemistry.chemical_classification Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) Chromatography Binding Sites Sequence Homology Amino Acid Fructose Hydrogen-Ion Concentration Peptide Fragments Rats Glutamine Dithiothreitol Kinetics Enzyme chemistry Liver Sequence Alignment |
| Zdroj: | Archives of biochemistry and biophysics. 379(2) |
| ISSN: | 0003-9861 |
| Popis: | 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 KM 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 Kinact of 5.0 μM. 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). |
| Databáze: | OpenAIRE |
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