PMID:3297136
| Citation |
Badet, B, Vermoote, P, Haumont, PY, Lederer, F and LeGoffic, F (1987) Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location. Biochemistry 26:1940-8 |
|---|---|
| Abstract |
L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., & Roseman, S. (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., & Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation. |
| Links | |
| Keywords |
Amino Acids/analysis; Binding Sites; Escherichia coli/enzymology; Glutamine/metabolism; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/isolation & purification; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism; Kinetics; Molecular Weight; Protein Binding; Sulfhydryl Reagents/pharmacology; Transaminases/isolation & purification |
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