title: A fluorescent peptide substrate for the surface metalloprotease of Leishmania.
abstract: A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.
authors.affiliation: Department of Pathology, School of Medicine, University of California, San Francisco 94143.
authors.forename: J
authors.initials: J
authors.lastname: Bouvier
authors.forename: P
authors.initials: P
authors.lastname: Schneider
authors.forename: B
authors.initials: B
authors.lastname: Malcolm
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D007891:Leishmania / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania