title: Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.
abstract: Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.
authors.affiliation: Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM, URA D1500 CNRS, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Marie-Claire
authors.forename: E
authors.initials: E
authors.lastname: Ruffet
authors.forename: S
authors.initials: S
authors.lastname: Antonczak
authors.forename: A
authors.initials: A
authors.lastname: Beaumont
authors.forename: M
authors.initials: M
authors.lastname: O'Donohue
authors.forename: B P
authors.initials: BP
authors.lastname: Roques
authors.forename: M C
authors.initials: MC
authors.lastname: Fournié-Zaluski
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics* / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001667:Binding, Competitive; D018076:DNA, Complementary / Q000235:genetics; D006020:Glycopeptides / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D013820:Thermolysin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015244:Thiorphan / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D006020:Glycopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D001120:Arginine; D015244:Thiorphan; D015260:Neprilysin; D013820:Thermolysin; C008890:phosphoramidon