Substrate Specificity at the P1´ Site of Escherichia coli OmpT under Denaturing Conditions

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  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 Arg¹⁴⁰-Arg¹⁴¹ and the modified substrates PRX substituted all 19 natural amino acids at the P1´ site instead of Arg¹⁴¹. 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 Arg¹⁴⁰ except for the Arg¹⁴⁰-Asp¹⁴¹, -Glu¹⁴¹, and -Pro¹⁴¹ pairs. In addition to Arg¹⁴⁰ at the P1 site, similar results were obtained when Lys¹⁴⁰ 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.