A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: competition kinetics as a tool for enzyme specificity investigation.
Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate's N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.
Evnouchidou
Irini
I
IRRP, National Centre for Scientific Research "Demokritos," Athens, Greece.
Berardi
Marcelo J
MJ
Stratikos
Efstratios
E
D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004140:Dinitrophenols / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D016018:Least-Squares Analysis; D045726:Metalloproteases / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D008954:Models, Biological; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D014364:Tryptophan / Q000032:analysis
D004140:Dinitrophenols; D005456:Fluorescent Dyes; D015778:Minor Histocompatibility Antigens; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014364:Tryptophan; D045726:Metalloproteases; D000626:Aminopeptidases; C464841:ERAP1 protein, human