Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2

Anal Biochem. 2003 Jan 15;312(2):141-7. doi: 10.1016/s0003-2697(02)00461-x.


Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.

MeSH terms

  • Carboxypeptidases / metabolism*
  • Enzyme Inhibitors
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • Humans
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Myocardium / enzymology
  • Nitric Oxide / chemistry
  • Peptides / chemistry*
  • Peptides / metabolism*
  • Peptidyl-Dipeptidase A
  • Phenylalanine / metabolism
  • Substrate Specificity
  • Tyrosine / metabolism


  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Peptides
  • Nitric Oxide
  • Tyrosine
  • Phenylalanine
  • Carboxypeptidases
  • Peptidyl-Dipeptidase A
  • angiotensin converting enzyme 2