N-bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11.
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 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa 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.
Bateman
R C
RC
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Kim
Y A
YA
Slaughter
C
C
Hersh
L B
LB
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D004151:Dipeptides / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D004743:Enkephalin, Leucine / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015260:Neprilysin / Q000037:antagonists & inhibitors* / Q000235:genetics; D015345:Oligonucleotide Probes; D051381:Rats
D000345:Affinity Labels; D004151:Dipeptides; D015345:Oligonucleotide Probes; C064274:N-bromoacetylleucylglycine; D004743:Enkephalin, Leucine; D015260:Neprilysin