Substrate Specificity of the Integral Membrane Protease OmpT Determined by Spatially Addressed Peptide Libraries

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Abstract
Escherichia coli outer membrane protease T (OmpT) is an endopeptidase that specifically cleaves between two consecutive basic residues. In this study we have investigated the substrate specificity of OmpT using spatially addressed SPOT peptide libraries. The peptide acetyl-Dap(dnp)-Ala-Arg↓Arg-Ala-Lys(Abz)-Gly was synthesized directly onto cellulose membrane. The peptide contained the aminobenzoyl (Abz) fluorophore, which was internally quenched by the dinitrophenyl (dnp) moiety. Treatment of the SPOT membrane with the small, water-soluble protease trypsin resulted in highly fluorescent peptide SPOTs. However, no peptide cleavage was observed after incubation with detergent-solubilized OmpT, a macromolecular complex with an estimated molecular mass of 180 kDa. This problem could be solved by the introduction of a long, polar polyoxyethylene glycol linker between the membrane support and the peptide. Peptide libraries for the P2, P1, P1‘, and P2‘ positions in the substrate were screened with OmpT, and peptides of positive SPOTs were resynthesized and subjected to kinetic measurements in solution. The best substrate Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly had a turnover number kcat of 40 s-1, which is 12-fold higher than the starting substrate. Peptides containing an acidic residue at P2 or P2‘ were not substrates for OmpT, suggesting that long-range electrostatic interactions are important for the formation of the enzyme−substrate complex. OmpT was highly selective toward l-amino acids at P1 but was less so at P1‘ where a peptide with d-Arg at P1‘ was a competitive inhibitor (Ki of 19 μM). An affinity chromatography resin based on these findings was developed, which allowed for the one-step purification of OmpT from a bacterial lysate. The implications of the determined consensus substrate sequence (Arg/Lys)↓(Arg/Lys)-Ala for the proposed biological function of OmpT in defense against antimicrobial peptides are discussed.