Active site-directed inactivation of Escherichia coli glucosamine-6-phosphate synthase. Determination of the fructose 6-phosphate binding constant using a carbohydrate-based inactivator.
Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. N-Iodoacetylglucosamine 6-phosphate is an active site-directed irreversible inactivator of GlmS from Escherichia coli (kinact/KI = 17 (+/-3) m-1 s-1). Both fructose 6-phosphate and glutamine protect the enzyme from inactivation, indicating that this reagent is directed at both the sugar binding site and the glutamine binding site. Protection studies with fructose 6-phosphate demonstrate that the value of the dissociation constant for fructose 6-phosphate is 3.3 (+/-0.5) x 10(-7) m, approximately 3 orders of magnitude less than the Kia value for this substrate determined from initial velocity experiments (Badet, B., Vermoote, P., and Le Goffic, F. (1988) Biochemistry 27, 2282-2287).
Bearne
S L
SL
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates / Q000378:metabolism*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D015394:Molecular Structure
D005636:Fructosephosphates; C027618:fructose-6-phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D000117:Acetylglucosamine