Development of an internally quenched fluorescent substrate for Escherichia coli leader peptidase.
Escherichia coli leader peptidase, an integral membrane protein, is responsible for the cleavage of the signal sequence of many exported proteins. Recent studies suggest that it is a novel serine protease that utilizes a serine-lysine catalytic dyad. In an effort to further understand 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(Abz) (anthraniloyl), was designed and synthesized. In the intact peptide, the fluorescence of the anthraniloyl group is quenched by the 3-nitrotyrosine. This quenched fluorescence is liberated upon cleavage of the 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 products 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 microM) and the kcat/K(m) was calculated to be 71.1 M-1 s-1. These data are comparable with the unmodified peptide substrate. This report represents the first direct continuous assay based on fluorescence resonance energy transfer for E. coli leader peptidase.
Zhong
W
W
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Benkovic
S J
SJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D009842:Oligopeptides; D012697:Serine Endopeptidases; C026515:type I signal peptidase