Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location.
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.
Badet
B
B
Vermoote
P
P
Haumont
P Y
PY
Lederer
F
F
LeGoffic
F
F
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000378:metabolism*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D000637:Transaminases / Q000302:isolation & purification*
D000596:Amino Acids; D013439:Sulfhydryl Reagents; D005973:Glutamine; D000637:Transaminases; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)