Substrate specificity of the integral membrane protease OmpT determined byspatially addressed peptide libraries

Citation
N. Dekker et al., Substrate specificity of the integral membrane protease OmpT determined byspatially addressed peptide libraries, BIOCHEM, 40(6), 2001, pp. 1694-1701
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
40
Issue
6
Year of publication
2001
Pages
1694 - 1701
Database
ISI
SICI code
0006-2960(20010213)40:6<1694:SSOTIM>2.0.ZU;2-8
Abstract
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.