Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant

R. C. Bateman, D. Jackson, Clive A. Slaughter, S. Unnithan, Y. G. Chai, C. Moomaw, L. B. Hersh

Research output: Contribution to journalArticle

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Abstract

Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.

Original languageEnglish (US)
Pages (from-to)6151-6157
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number11
StatePublished - Jan 1 1989
Externally publishedYes

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Neprilysin
Glutamine
Arginine
Rats
Catalytic Domain
Enkephalins
Phenylglyoxal
Amides
High pressure liquid chromatography
Peptides
Substrates
Enzymes
Digestion
phenylalanylalanine
phenylalanylglycine
alanylglycine
High Pressure Liquid Chromatography
Mutagenesis
Pepsin A
Site-Directed Mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant. / Bateman, R. C.; Jackson, D.; Slaughter, Clive A.; Unnithan, S.; Chai, Y. G.; Moomaw, C.; Hersh, L. B.

In: Journal of Biological Chemistry, Vol. 264, No. 11, 01.01.1989, p. 6151-6157.

Research output: Contribution to journalArticle

Bateman, R. C. ; Jackson, D. ; Slaughter, Clive A. ; Unnithan, S. ; Chai, Y. G. ; Moomaw, C. ; Hersh, L. B. / Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 11. pp. 6151-6157.
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abstract = "Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.",
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