ACTIVE-SITE-DIRECTED INACTIVATION OF ESCHERICHIA-COLI GLUCOSAMINE-6-PHOSPHATE SYNTHASE - DETERMINATION OF THE FRUCTOSE 6-PHOSPHATE BINDING CONSTANT USING A CARBOHYDRATE-BASED INACTIVATOR
Sl. Bearne, ACTIVE-SITE-DIRECTED INACTIVATION OF ESCHERICHIA-COLI GLUCOSAMINE-6-PHOSPHATE SYNTHASE - DETERMINATION OF THE FRUCTOSE 6-PHOSPHATE BINDING CONSTANT USING A CARBOHYDRATE-BASED INACTIVATOR, The Journal of biological chemistry, 271(6), 1996, pp. 3052-3057
Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glu
cosamine B-phosphate from fructose 6-phosphate using glutamine as the
ammonia source, Because N-acetylglucosamine is an essential building b
lock of both bacterial cell walls and fungal cell wall chitin, the enz
yme is a potential target for antibacterial and antifungal agents, N-I
odoacetylglucosamine 6-phosphate is an active site-directed irreversib
le inactivator of GlmS from Escherichia coli (k(inact)/K-I = 17 (+/-3)
M(-1) s(-1)). Both fructose 6-phosphate and glutamine protect the enz
yme from inactivation, indicating that this reagent is directed at bot
h the sugar binding site and the glutamine binding site, Protection st
udies with fructose g-phosphate demonstrate that the value of the diss
ociation constant for fructose 6-phosphate is 3.3 (+/-0.5) x 10(-7) M,
approximately 3 orders of magnitude less than the K-ia value for this
substrate determined from initial velocity experiments (Badet, B., Ve
rmoote, P., and Le Goffic, F. (1988) Biochemistry 27, 2282-2287).