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Kinetic measurements on single-molecule disulfide bond cleavage.

ABSTRACT: We use single-molecule force clamp spectroscopy (SMFCS) to explore the reactivity of tris(2-carboxyethyl)phosphine (TCEP), 1, 4-dl-dithiothreitol (DTT) and hydrosulfide anion (HS(-)) on disulfide bonds within a mechanically stretched polypeptide. The single-bond level bimolecular nucleophilic substitution (S(N)2) events are recorded at a series of precisely controlled temperatures so that the Arrhenius kinetic parameters, that is, the height of the activation energy barrier (E(a)) and the attempting frequency (A) of the chemical reactions, can be determined. The values of A are typically at the order of 10(7) M(-1) s(-1), which is far lower than that predicted by the transition-state theory, in which A is given by k(B)T/h and around 10(12) M(-1) s(-1) at room temperature. Furthermore, E(a) is derived to be 30-40 kJ/mol, which can be lowered by ?6-8% with every 100 pN mechanical force applied. The correlation of the A and E(a) with the molecular structures reveals that the relative magnitude of these two parameters cannot be simply judged from the size of the molecule or the nucleophilicity of the attacking atom. The comparison of the influences on the reaction rate induced by force and temperature indicates an equivalent accelerating effect by every 50 pN or 10 K increment, giving for the first time the relationship between mechanical and thermal effects on a single-molecule S(N)2 chemical reaction.

SUBMITTER: Liang J

PROVIDER: S-EPMC3711696 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Kinetic measurements on single-molecule disulfide bond cleavage.

Liang Jian J Fernández Julio M JM

Journal of the American Chemical Society 20110222 10

We use single-molecule force clamp spectroscopy (SMFCS) to explore the reactivity of tris(2-carboxyethyl)phosphine (TCEP), 1, 4-dl-dithiothreitol (DTT) and hydrosulfide anion (HS(-)) on disulfide bonds within a mechanically stretched polypeptide. The single-bond level bimolecular nucleophilic substitution (S(N)2) events are recorded at a series of precisely controlled temperatures so that the Arrhenius kinetic parameters, that is, the height of the activation energy barrier (E(a)) and the attemp ...[more]

PMID: 21341766

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Project description:Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other's virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-amino-acid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicans IMPORTANCE Enterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge.

Dataset Information

Kinetic measurements on single-molecule disulfide bond cleavage.

Publications

Kinetic measurements on single-molecule disulfide bond cleavage.

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