IDENTIFICATION OF PEPTIDES INHIBITING ENZYME-I OF THE BACTERIAL PHOSPHOTRANSFERASE SYSTEM USING COMBINATORIAL CELLULOSE-BOUND PEPTIDE LIBRARIES

The phosphoenolpyruvate(P-pyruvate)-dependent sugar phosphotransferase system (PTS) is a transport and signal-transduction system which is a lmost ubiquitous in bacteria but does not occur in eucaryotes. It cata lyzes the uptake and phosphorylation of carbohydrates and is involved in signal transduction, e.g. catabolite repression, chemotaxis, and al losteric regulation of metabolic enzymes and transporters. EI (Enzyme I of the PTS) is the first and central component of the divergent PTS (P-pyruvate -dependent sugar phosphotransferase system) phosphorylatio n cascade. Using immobilized combinatorial peptide libraries and phosp horimaging, heptapeptides and octapeptides were identified which selec tively inhibit EI in vitro. The IC50 of the best peptides is 30 mu M w hich is close to the K-M (6 mu M) of EI for its natural substrate HPr (histidine containing phosphoryl carrier protein of the PTS). The affi nity-selected peptides are better inhibitors than a peptide with the a ctive-site sequence of HPr. The selected peptides contain several basi c residues and one aromatic residue which do not occur in the active s ite bf HPr. The large proportion of basic residues most likely reflect s charge complementarity to the strongly acidic active-site pocket of EI. Guanidino groups might facilitate by complexation of the phosphory l group the slow phosphorylation of the peptide.