AN AMINO-ACID SWITCH (GLY(281)ASTERISK-ARG) WITHIN THE HINGE REGION OF THE TRYPTOPHAN SYNTHASE BETA-SUBUNIT CREATES A NOVEL CLEAVAGE SITE FOR THE OMPT PROTEASE AND SELECTIVITY DIMINISHES AFFINITY TOWARD A SPECIFIC MONOCLONAL-ANTIBODY

The in vitro susceptibility to endogenous proteases of the beta subuni t of Escherichia coli tryptophan synthase was studied immunochemically . Whereas the wild-type beta subunit was apparently very stable, the m issense mutant beta(BS), carrying an amino acid switch from Gly to Arg at residue 281, underwent specific proteolytic cleavage. Polyclonal c hicken antibodies and monoclonal antibodies specific for the N terminu s (monoclonal antibody (mAb) 15-1), the C terminus (mAb 93-6), and the ''hinge'' region (mAb 164-2) were used to study the hydrolysis of the beta(B8) polypeptide. Cleavage products of 30 kDa, from the N terminu s, and 13 kDa, from the C terminus, were observed. These two polypepti des correspond to the well characterized F1 (N-terminal) and F2 (C-ter minal) fragments that are generated during the limited tryptic proteol ysis of the wild-type beta subunit. The outer membrane-associated prot ease OmpT was shown to be responsible for the cleavage of the beta(B8) mutant protein. Proteolytic cleavage, observed only under neutral non -denaturing conditions, was specific for the peptide bond between Arg2 81 and Met282. The Arg-Met peptide bond has not previously been report ed to be susceptible to cleavage by the OmpT protease. The beta(B8) po lypeptide had dramatically reduced affinity for mAb 164-2. This antibo dy interacted more strongly with the OmpT-generated F1-like fragment t han with the intact beta(B8) protein. These results strongly suggest t hat the G281R mutation alters the conformation of the hinge region of the mutant beta subunit, particularly the beta-turn around Gly281. The implications with respect to the epitope recognized by mAb 164-2 are discussed.