Navegando por Palavras-chave "Kex2"

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    Specificity characterization of the alpha-mating factor hormone by Kex2 protease
    (Elsevier France-Editions Scientifiques Medicales Elsevier, 2016) Manfredi, Marcella Araujo; Antunes, Alyne Alexandrino; Passos Jesus, Larissa de Oliveira; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; de Souza Judice, Wagner Alves
    Kex2 is a Ca2+-dependent serine protease from S. cerevisiae. Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S' of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR down arrow EADQ-EDDnp and Abz-YKR down arrow EADQ-EDDnp based on the pro-a-mating factor sequence. The specificity of Kex2 due to basic residues at P-1' is affected by the type of residue in the P-3 position. Some residues in P-1' with large or bulky side chains yielded poor substrate specificity. The k(cat)/K-M values for peptides with P-2' substitutions containing Tyr in P-3 were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in k(cat) and K-M, respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a poly peptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
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    Thermodynamic analysis of Kex2 activity: The acylation and deacylation steps are potassium- and substrate-dependent
    (Elsevier Science Bv, 2018) Antunes, Alyne Alexandrino; Jesus, Larissa de Oliveira Passos; Manfredi, Marcella Araujo; Souza, Aline Aparecida de; Machado, Mauricio Ferreira Marcondes; Silva, Pamela Moraes e; Icimoto, Marcelo Yudi [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Judice, Wagner Alves de Souza
    Kex2 is the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave at sites containing pairs of basic residues. Here, we studied the effects of KCl on the individual rate constants of association, dissociation, acylation and deacylation and determined the thermodynamic parameters at each step of the Kex2 reaction. Potassium bound Kex2 with K-D = 20.3 mM. The order in which potassium entered the reaction system modified the effect of activation or inhibition, which depended on the size of the substrate. A possible allosteric potassium binding site at the S-6 subsite was involved in activation, and a distant site located between the catalytic domain and the P-domain was involved in inhibition. Potassium decreased the energetic barriers of almost all steps of catalysis. The acylation of Ac-PMYKR-AMC in the absence of potassium was the rate-limiting step. Therefore, for substrates containing a P-1-Arg, the deacylation step is not necessarily the rate-limiting event, and other residues at the P' positions may participate in controlling the acylation and deacylation steps. Thus, it is reasonable to conclude that potassium is involved in the processing of the alpha-mating factor that promotes Ca2+, mobilization by activating a high-affinity Ca2+-influx system to increase the cytosolic [Ca2+], resulting in the activation of channels that are essential for the survival of Saccharomyces cerevisine cells.