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 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).