Navegando por Palavras-chave "enzyme specificity"

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    A comparative conformational analysis of thimet oligopeptidase (EC 3.4.24.15) substrates
    (Munksgaard Int Publ Ltd, 1998-06-01) Jacchieri, S. G.; Gomes, M. D.; Juliano, L.; Camargo, ACM; Fundacao Antonio Prudente; Inst Butantan; Universidade Federal de São Paulo (UNIFESP)
    The specificity of thimet oligopeptidase (EC 3.4.24.15) (TOP 24.15) does not agree with theoretical models devised to explain the specificity characteristic of peptidases toward certain sequences of amino acid residues. According to previous studies peptide chains hydrolyzed by TOP 24.15 adopt similar main chain conformations, although with different and in some cases small probabilities of occurrence in aqueous solution, To determine specific structural features recognized by TOP 24.15, a conformational search including eight polypeptides with known susceptibilities for catalytic hydrolysis was executed and the distribution of each main chain conformation found in the search was tabulated. Two sets of main chain conformations were selected, those common to all peptides in the study and those common only to substrates of TOP 24.15. The former set is very small and includes mainly extended conformations. In contrast, the latter set is large and its conformations are coiled and exhibit sharp turns coincident with positions of hydrolysis by TOP 24.15. These results indicate a possible basis for the selectivity of TOP 24.15. (C) Munksgaard 1998.
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    Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH-inducible fluorogenic substrate of trypanosomal cysteine proteinases
    (Blackwell Science Ltd, 1999-01-01) Serveau, C.; Lalmanach, G.; Hirata, Izaura Yoshico [UNIFESP]; Scharfstein, J.; Juliano, Maria Aparecida [UNIFESP]; Gauthier, F.; Univ Tours; Universidade Federal de São Paulo (UNIFESP); Universidade Federal do Rio de Janeiro (UFRJ)
    The substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, was investigated using a series of dansyl-peptides based on the putative autoproteolytic sequence of the proteinase (VVG-GP) located at the hinge region between the catalytic domain and the C-terminal extension. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cathepsin L cleaved substrate with a His at that position. the combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cruzipain was able to cleave the HPGGP peptide at the GG bond. A substrate with intramolecularly quenched fluorescence was raised on this sequence (Abz-HPGGPQ-EDDnp) which was also specifically cleaved by cruzipain (k(cat)/K-m of 157 000 (M-1.s-1)) and by a homologous proteinase from Trypanosoma congolense. the pH activity profile of cruzipain on Abz-HPGGPQ-EDDnp showed a narrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although trypanosomal cysteine proteinases remain active at basic pH. the lack of activity at neutral and basic pH was due to a decrease in k(cat), while the K-m remained essentially unchanged, demonstrating that the substrate still binds to the enzyme and therefore behaves as an inhibitor. Changing the substrate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz-HPGGPQ-EDDnp also inhibited mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. the importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. the resulting peptide was not cleaved by cruzipain in spite of the presence of a positively charged group at P3, but still interacted with the enzyme. It was concluded that the presence of an imidazolium group at P3 was essential to endow the HPGGPQ sequence with the properties of a cruzipain substrate.
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    New insights into the substrate specificity of macrophage elastase MMP-12
    (Walter De Gruyter Gmbh, 2016) Lamort, Anne-Sophie; Gravier, Rodolphe; Laffitte, Anni; Juliano, Luiz [UNIFESP]; Zani, Marie-Louise; Moreau, Thierry
    Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence ProX-X down arrow Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.
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    Proteinase inhibition using small Bowman-Birk-type structures
    (Funpec-editora, 2007-01-01) Fernandez, J. H.; Mello, M. O.; Galgaro, L.; Tanaka, Aparecida Sadae [UNIFESP]; Silva-Filho, M. C.; Neshich, G.; Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA); Universidade Federal de São Paulo (UNIFESP); Escuela Super Agr Luiz de Queiroz; LNCC
    Bowman-Birk inhibitors (BBIs) are cysteine-rich and highly cross-linked small proteins that function as specific pseudosubstrates for digestive proteinases. They typically display a double-headed structure containing an independent proteinase-binding loop that can bind and inhibit trypsin, chymotrypsin and elastase. In the present study, we used computational biology to study the structural characteristics and dynamics of the inhibition mechanism of the small BBI loop expressing a 35-amino acid polypeptide (ChyTB2 inhibitor) which has coding region for the mutated chymotrypsin-inhibitory site of the soybean BBI. We found that in the BBI-trypsin inhibition complex, the most important interactions are salt bridges and hydrogen bonds, whereas in the BBI-chymotrypsin inhibition complex, the most important interactions are hydrophobic. At the same time, ChyTB2 mutant structure maintained the individual functional domain structure and excellent binding/ inhibiting capacities for trypsin and chymotrypsin at the same time. These results were confirmed by enzyme-linked immunosorbend assay experiments. The results showed that modeling combined with molecular dynamics is an efficient method to describe, predict and then obtain new proteinase inhibitors. For such study, however, it is necessary to start from the sequence and structure of the mutant interacting relatively strongly with both trypsin and chymotrypsin for designing the small BBI-type inhibitor against proteinases.
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    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.