Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

The Biochemical Journal
Katherine PaschalidouChryssa Tzougraki

Abstract

The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3' (substrate 3) resulted in an acceptable k(cat)/K(m) (41000 M(-1).s(-1)) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a k(cat)/K(m) value of 350000 M(-1).s(-1) and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for k(cat). The pH dependence of k(cat)/K(m) for renin and substrate 9 showed that the optimal pH is at pH 6-7. It also showed two titrating group...Continue Reading

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Citations

Jun 15, 2006·Analytical Chemistry·Kristin A FletcherIsiah M Warner
Jun 8, 2012·American Journal of Physiology. Renal Physiology·Jun ZhangJeffrey D Peterson
Apr 20, 2011·Analytical and Bioanalytical Chemistry·Francesca ManciniVincenza Andrisano
Aug 14, 2009·Journal of Pharmaceutical and Biomedical Analysis·Francesca Mancini, Vincenza Andrisano
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Jul 3, 2019·International Journal of Molecular Sciences·Ryan D SullivanInna P Gladysheva
May 15, 2020·Biochimica Et Biophysica Acta. Proteins and Proteomics·Julian BreidenbachMichael Gütschow
Sep 26, 2012·Analytical Chemistry·Yi LongChun-yang Zhang

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