Navegando por Palavras-chave "catalytic mechanism"

Agora exibindo 1 - 2 de 2
  • Nenhuma Miniatura disponível
    Item
    Somente Metadadados
    BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs
    (Elsevier B.V., 2008-04-01) Nascimento-Silva, Maria Clara L.; Leal, Alexandre T.; Daffre, Sirlei; Juliano, Luiz [UNIFESP]; Silva Vaz, Itabajara da; Paiva-Silva, Gabriela de O.; Oliveira, Pedro L.; Sorgine, Marcos Henrique F.; Universidade Federal do Rio de Janeiro (UFRJ); Univ Fed Rio Grande do Sul; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., de Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W, Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525-532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, VI., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66,331-341]. in this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. in spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high beta sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae. (c) 2007 Elsevier Inc. All rights reserved.
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis
    (Elsevier B.V., 2003-04-24) Oliveira, Vitor [UNIFESP]; Araujo, M. C.; Rioli, V; Camargo, Antonio Carlos Martins de [UNIFESP]; Tersariol, Ivarne Luis dos Santos [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Ferro, Emer Suavinho [UNIFESP]; Univ Mogi das Cruzes; Inst Butantan; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    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. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.