Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties.
The proteolysis of native glucosamine-6-phosphate synthase (Mr 67,000) from Escherichia coli was investigated using two nonspecific and five specific endoproteinases, alpha-chymotrypsin generated two nonoverlapping polypeptides CT1 and CT2 of Mr 40,000 and 27,000 lacking glucosamine-6P synthesizing activity. Amino terminal and carboxy terminal sequence analysis showed that cleavage occurred between positions 240 and 241 of the primary sequence without further degradation. The glutamine amidohydrolase activity was located in the CT2 N-terminal polypeptide which was capable of incorporating 0.7 equivalent of the glutamine site-directed affinity label [2-3H]-N3-(4-methoxyfumaroyl)-diaminopropionic acid indicating that it bears the amidotransferase function. CT1 which displayed a higher reactivity than CT2 for fructose-6P binding contains the ketose/aldose isomerase activity. These data suggest the existence of a hinge structure essential for the catalytically efficient coupling between the ammonia generating domain and the sugar binding domain and support the model recently proposed by Mei and Zalkin in which purF-type amidotransferases contain a glutamine hydrolase domain of approximately 200 amino acids fused to an ammonia-transfer domain.
Denisot
M A
MA
Laboratoire de Bioorganique et Biotechnologies, UA CNRS 1389, Paris, France.
Le Goffic
F
F
Badet
B
B
D000345:Affinity Labels; D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates; D005973:Glutamine; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D010447:Peptide Hydrolases
D000345:Affinity Labels; D005636:Fructosephosphates; D005973:Glutamine; C027618:fructose-6-phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D010447:Peptide Hydrolases; D000581:Amidohydrolases; D002238:Carbohydrate Epimerases