EPITOPE MAPPING AND TIGHT-BINDING INHIBITION WITH MONOCLONAL-ANTIBODIES DIRECTED AGAINST ESCHERICHIA-COLI GLUCOSAMINE 6-PHOSPHATE SYNTHASE

In the present work, we attempt to identify inhibitory monoclonal anti bodies directed against Escherichia coli glucosamine-6P synthase (GlmS ) and to localize the corresponding epitopes to better understand the topology of the enzyme during catalysis. Four of the 15 monoclonal ant ibodies have been shown to be specific for the native form of the enzy me and 2 of them, 505,1 and 522.2, strongly inhibit the glucosamine sy nthase activity, Kinetic analysis of 505.1 antibody behavior revealed a tight-binding inhibition with a K-i = 40 +/- 20 pM, a value which is four orders of magnitude lower than the best active site-directed inh ibitor reported so far. The reactivity of all the monoclonal antibodie s with 601 overlapping octapeptides covering the entire sequence of Gl mS was tested by enzyme-linked immunosorbent assay for precise epitope mapping, Four linear epitopes specific for the denatured protein and one present in both native and denatured enzyme were defined by this a pproach, Neither 505.1 nor 522.2 was directed against linear epitopes, However, evidence for the binding of 505.1 at the glutamine catalytic site was shown by using site-directed mutants of GlmS as well as by c ompetition experiments with an irreversible inhibitor, The mAb 105.1, which recognizes the octapeptide containing the sequence RWATHG conser ved among the six glucosamine-6P synthases reported so far, allowed th e detection of the human enzyme. (C) 1995 Academic Press, Inc.