N-Bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11

R. C. Bateman, Y. A. Kim, Clive A. Slaughter, L. B. Hersh

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37°C. The concentration dependence of inactivation revealed saturation kinetics with an apparent K(i) of 10 mM and k(inact) of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue.

Original languageEnglish (US)
Pages (from-to)8365-8368
Number of pages4
JournalJournal of Biological Chemistry
Volume265
Issue number15
StatePublished - Jun 12 1990
Externally publishedYes

Fingerprint

glycylleucine
Affinity Labels
Neprilysin
Enzymes
Histidine
phenylalanylglycine
Mutagenesis
Kinetics
Enkephalins
High performance liquid chromatography
Site-Directed Mutagenesis
Glutamine
Digestion
Catalytic Domain
High Pressure Liquid Chromatography
Peptides
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

N-Bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11. / Bateman, R. C.; Kim, Y. A.; Slaughter, Clive A.; Hersh, L. B.

In: Journal of Biological Chemistry, Vol. 265, No. 15, 12.06.1990, p. 8365-8368.

Research output: Contribution to journalArticle

Bateman, R. C. ; Kim, Y. A. ; Slaughter, Clive A. ; Hersh, L. B. / N-Bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 15. pp. 8365-8368.
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