DEVELOPMENT OF AN INTERNALLY QUENCHED FLUORESCENT SUBSTRATE FOR ESCHERICHIA-COLI LEADER PEPTIDASE

Escherichia coli leader peptidase, an integral membrane protein, is re sponsible for the cleavage of the signal sequence of many exported pro teins. Recent studies suggest that it is a novel serine protease that utilizes a serine-lysine catalytic dyad. In an effort to further under stand the mechanism of this enzyme, an internally quenched fluorescent peptide substrate incorporating the leader peptidase cleavage site of maltose binding protein signal peptide, Y(NO2)-F-S-A-S-A-L-A-K-I-K(Ab z) (anthraniloyl), was designed and synthesized. In the intact peptide , the fluorescence of the anthraniloyl group is quenched by the 3-nitr otyrosine. This quenched fluorescence is Liberated upon cleavage of th e peptide by the leader peptidase, resulting in increased fluorescence that could then be monitored fluorometrically. The designed substrate can be cleaved effectively by E. coli leader peptidase as detected by both HPLC and fluorescent spectroscopy. Mass spectra of cleavage prod ucts demonstrated that the cleavage occurs at the predicted site (A-K) . The cleavage of the peptide substrate has a Linear dependence on the enzyme concentration (0.1 to 1.9 mu M) and the k(cat)/K-m was calcula ted to be 71.1 M-1 s(-1). These data are comparable with the unmodifie d peptide substrate. This report represents the first direct continuou s assay based on fluorescence resonance energy transfer for E. coli le ader peptidase. (C) 1998 Academic Press.