N. Dekker et al., Substrate specificity of the integral membrane protease OmpT determined byspatially addressed peptide libraries, BIOCHEM, 40(6), 2001, pp. 1694-1701
Escherichia coli outer membrane protease T (OmpT) is an endopeptidase that
specifically cleaves between two consecutive basic residues. Ln this study
we have investigated the substrate specificity of OmpT using spatially addr
essed SPOT peptide libraries. The peptide acetyl-Dap(dnp)-Ala-Arg down arro
w Arg-Ala-Lys(Abz)-Gly was synthesized directly onto cellulose membrane. Th
e peptide contained the aminobenzoyl (Abz) fluorophore, which was internall
y quenched by the dinitrophenyl (dnp) moiety. Treatment of the SPOT membran
e with the small, water-soluble protease trypsin resulted in highly fluores
cent peptide SPOTs. However, no peptide cleavage was observed after incubat
ion with detergent-solubilized OmpT, a macromolecular complex with an estim
ated molecular mass of 180 kDa. This problem could be solved by the introdu
ction of a long, polar polyoxyethylene glycol linker between the membrane s
upport and the peptide. Peptide libraries for the P-2, P-1, P-1', and P-2'
positions in the substrate were screened with OmpT, and peptides of positiv
e SPOTs were resynthesized and subjected to kinetic measurements in solutio
n. The best substrate Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly had a turnover numbe
r k(cat) of 40 s(-1) which is 12-fold higher than the starting substrate. P
eptides containing an acidic residue at P1 or P-2' were not substrates for
OmpT, suggesting that long-range electrostatic interactions are important f
or the formation of the enzyme-substrate complex. OmpT was highly selective
toward L-amino acids at PI but was less so at P-1' where a peptide with D-
Arg at P-1' was a competitive inhibitor (Ki of 19 muM) An affinity chromato
graphy resin based on these findings was developed, which allowed for the o
ne-step purification of OmpT from a bacterial lysate. The implications of t
he determined consensus substrate sequence (Arg/ Lys)down arrow (Arg/Lys)-A
la for the proposed biological function of OmpT in defense against antimicr
obial peptides are discussed.