RESUMO
The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K(D)=7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.
Assuntos
Antígenos de Dermatophagoides/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Dobramento de Proteína , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes , Dicroísmo Circular , Simulação por Computador , Cisteína Endopeptidases , Dermatophagoides pteronyssinus/enzimologia , Dermatophagoides pteronyssinus/imunologia , Precursores Enzimáticos , Fluorescência , Glicosilação , Concentração de Íons de Hidrogênio , Pichia/genética , Pichia/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes , Ressonância de Plasmônio de SuperfícieRESUMO
The role of the mobile loop comprising residues 60-66 in metallo-beta-lactamases has been studied by site-directed mutagenesis, determination of kinetic parameters for six substrates and two inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and molecular modeling. The W64A mutation was performed in IMP-1 and BcII (after replacement of the BcII 60-66 peptide by that of IMP-1) and always resulted in increased K(i) and K(m) and decreased k(cat)/K(m) values, an effect reinforced by complete deletion of the loop. k(cat) values were, by contrast, much more diversely affected, indicating that the loop does not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand is also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications.