title: A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis.
abstract: Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, Brazil. vitor.biof@infar.epm.br
authors.forename: Vitor
authors.initials: V
authors.lastname: Oliveira
authors.forename: Maurício C
authors.initials: MC
authors.lastname: Araújo
authors.forename: Vanessa
authors.initials: V
authors.lastname: Rioli
authors.forename: Antonio C M
authors.initials: AC
authors.lastname: de Camargo
authors.forename: Ivarne L S
authors.initials: IL
authors.lastname: Tersariol
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Emer S
authors.initials: ES
authors.lastname: Ferro
mesh_terms: D000409:Alanine / Q000235:genetics / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics / Q000502:physiology
chemical_list: D014443:Tyrosine; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; C062316:neurolysin; D000409:Alanine