A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid.
作者信息
Knight C G
机构信息
Department of Biochemistry, Strangeways Research Laboratory, Cambridge, U.K.
出版信息
Biochem J. 1991 Feb 15;274 ( Pt 1)(Pt 1):45-8. doi: 10.1042/bj2740045.
Abstract
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.
摘要
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