New substrates for enkephalinase (neutral endopeptidase) based on fluorescence energy transfer.
Novel fluorescent substrates for enkephalinase (neutral endopeptidase; EC 3.4.24.11) have been developed. These new assays are based on the disappearance of energy transfer between a tryptophan or a tyrosine residue and the 5-dimethylaminonaphthalene-1-sulfonyl group (dansyl) in the substrates dansyl-Gly-Trp-Gly or dansyl-Gly-Tyr-Gly upon hydrolysis of their Gly-Trp or Gly-Tyr amide bond by enkephalinase. No significant difference in Km or kcat values were found for dansyl-Gly-Trp-Gly and dansyl-Gly-Tyr-Gly, indicating that, in contrast to thermolysin, the active site of enkephalinase easily accommodates tryptophan residues. Both tryptophan and tyrosine-containing substrates can be used for continuous recording of enkephalinase activity and should prove useful for detailed study of the substrate specificity of this enzyme.
D000818:Animals; D003619:Dansyl Compounds / Q000138:chemical synthesis; D010450:Endopeptidases / Q000378:metabolism*; D004735:Energy Transfer; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D015260:Neprilysin; D009842:Oligopeptides / Q000138:chemical synthesis; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D003619:Dansyl Compounds; D009842:Oligopeptides; D010450:Endopeptidases; D015260:Neprilysin