Substrate specificity at the P1' site of Escherichia coli ompT under denaturing conditions

Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg sup140 - Arg sup141 and the modified substrates PRX substituted all 19 natural amino acids at the P1' site instead of Arg sup141. OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg sup140 except for the Arg sup140 - Asp sup141, -Glu sup141, and -Pro sup141 pairs. In addition to Arg sup140 at the P1 site, similar results were obtained when Lys sup140 was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1' site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.