title: Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis. Histidine residues 583 and 587 are essential for catalysis.
abstract: Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His-142 and His-146) and a third histidine (His-231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His-583, His-587 and His-637) was explored by site-directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS-1 cells. Substitution of either His-583 or His-587 of NEP for Phe completely abolished the activity and Zn-directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His-142 and His-146 of thermolysin as zinc ligands. In contrast, substitution of His-637 for a phenylalanine residue was without effect on enzyme activity.
authors.affiliation: Département de biochimie, Université de Montréal, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Devault
authors.forename: V
authors.initials: V
authors.lastname: Sales
authors.forename: C
authors.initials: C
authors.lastname: Nault
authors.forename: A
authors.initials: A
authors.lastname: Beaumont
authors.forename: B
authors.initials: B
authors.lastname: Roques
authors.forename: P
authors.initials: P
authors.lastname: Crine
authors.forename: G
authors.initials: G
authors.lastname: Boileau
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D015260:Neprilysin
chemical_list: D000911:Antibodies, Monoclonal; D006639:Histidine; D004247:DNA; D008666:Metalloendopeptidases; D015260:Neprilysin