A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid.
DL-2-Amino-3-(7-methoxy-4-coumaryl)propionic acid, a new fluorescent amino acid (abbreviated to Amp), has been synthesized to provide an alternative to tryptophan in quenched fluorescent peptide substrates for peptidases. The model compound Ac-DL-Amp-NH2 was intensely fluorescent with an excitation maximum at 328 nm and an emission maximum at 392 nm. Fmoc (fluoren-9-ylmethoxycarbonyl)-DL-Amp was made to allow the solid-phase synthesis of Amp-containing peptides by the Fmoc-polyamide method. The peptide derivative Dnp (2,4-dinitrophenyl)-Pro-Leu-Gly-Pro-DL-Amp-D-Lys was cleaved by thimet peptidase at the Leu-Gly bond, with a 20-fold enhancement of fluorescence. The value of kcat./Km for thimet peptidase was 6.7 x 10(5) M-1.s-1, compared with the value of 2.4 x 10(5) M-1.s-1 for the tryptophan-containing analogue, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys.
Knight
C G
CG
Department of Biochemistry, Strangeways Research Laboratory, Cambridge, U.K.
D000409:Alanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D000595:Amino Acid Sequence; D003374:Coumarins / Q000138:chemical synthesis*; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D003374:Coumarins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009842:Oligopeptides; C067610:2-amino-3-(7-methoxy-4-coumaryl)propionic acid; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D000409:Alanine