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Mechanistic pathways of mercury removal from the organomercurial lyase active site.

Silva PJ, Rodrigues V - PeerJ (2015)

Bottom Line: Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+).Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction.Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

View Article: PubMed Central - HTML - PubMed

Affiliation: FP-ENAS/Fac. de Ciências da Saúde, Universidade Fernando Pessoa , Porto , Portugal.

ABSTRACT
Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

No MeSH data available.


Addition of deprotonated thiol to MerB-bound Hg2+.(A) Thiolate-based Int1; (B) breaking the Cys159-Hg bond (transition state); (C) thiolate-based Int2 (C96-bound); (D) breaking the Cys96-Hg bond (transition state); (E) thiolate-based Int2 (C159-bound). Molecules (D–E) are depicted as seen from a point of view approximately opposite that used in the depiction of molecules (A–C). Relevant distances (in Ångstrom) are highlighted.
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fig-4: Addition of deprotonated thiol to MerB-bound Hg2+.(A) Thiolate-based Int1; (B) breaking the Cys159-Hg bond (transition state); (C) thiolate-based Int2 (C96-bound); (D) breaking the Cys96-Hg bond (transition state); (E) thiolate-based Int2 (C159-bound). Molecules (D–E) are depicted as seen from a point of view approximately opposite that used in the depiction of molecules (A–C). Relevant distances (in Ångstrom) are highlighted.

Mentions: In contrast to the addition of cysteine thiols analyzed above, addition of a cysteine thiolate to the MerB-bound Hg2+ (Fig. 4) proceeds unhindered, i.e., without any energetic barrier. The tetra-coordinated intermediate formed (Int1) lies 12–13 kcal mol−1 below the infinitely-separated reactants (in MP2; 6–7 kcal mol−1 below reactants in DFT), and may then shed any of its Cys-ligands upon overcoming a moderate 14.0–15.5 kcal mol−1 barrier. The addition of a second thiolate to this complex, however, is much costlier due to the electrostatic repulsion between the freed, deprotonated, Cys and the negatively-charged thiolate. The precise cost depends very steeply on the chosen dielectric constant (Table 3), as expected for a reaction involving highly localized charges, but the transition state for this step always remains more than 25 kcal mol−1 above Int1, far above the 16–20 kcal mol−1 expected (Parks et al., 2009) for the rate-limiting step of this enzyme from the application of the Eyring equation, , to the experimentally observed reaction rate (Begley, Walts & Walsh, 1986b). Since methanethiol is weakly acidic (pKa = 10.4) and is almost completely protonated at physiological pH, the initial cost of deprotonating it must be added to the computations above. This cost amounts to −RT ln 10pH-pKa (Ullmann & Knapp, 1999), or 4.6 kcal mol−1 at pH = 7.


Mechanistic pathways of mercury removal from the organomercurial lyase active site.

Silva PJ, Rodrigues V - PeerJ (2015)

Addition of deprotonated thiol to MerB-bound Hg2+.(A) Thiolate-based Int1; (B) breaking the Cys159-Hg bond (transition state); (C) thiolate-based Int2 (C96-bound); (D) breaking the Cys96-Hg bond (transition state); (E) thiolate-based Int2 (C159-bound). Molecules (D–E) are depicted as seen from a point of view approximately opposite that used in the depiction of molecules (A–C). Relevant distances (in Ångstrom) are highlighted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525700&req=5

fig-4: Addition of deprotonated thiol to MerB-bound Hg2+.(A) Thiolate-based Int1; (B) breaking the Cys159-Hg bond (transition state); (C) thiolate-based Int2 (C96-bound); (D) breaking the Cys96-Hg bond (transition state); (E) thiolate-based Int2 (C159-bound). Molecules (D–E) are depicted as seen from a point of view approximately opposite that used in the depiction of molecules (A–C). Relevant distances (in Ångstrom) are highlighted.
Mentions: In contrast to the addition of cysteine thiols analyzed above, addition of a cysteine thiolate to the MerB-bound Hg2+ (Fig. 4) proceeds unhindered, i.e., without any energetic barrier. The tetra-coordinated intermediate formed (Int1) lies 12–13 kcal mol−1 below the infinitely-separated reactants (in MP2; 6–7 kcal mol−1 below reactants in DFT), and may then shed any of its Cys-ligands upon overcoming a moderate 14.0–15.5 kcal mol−1 barrier. The addition of a second thiolate to this complex, however, is much costlier due to the electrostatic repulsion between the freed, deprotonated, Cys and the negatively-charged thiolate. The precise cost depends very steeply on the chosen dielectric constant (Table 3), as expected for a reaction involving highly localized charges, but the transition state for this step always remains more than 25 kcal mol−1 above Int1, far above the 16–20 kcal mol−1 expected (Parks et al., 2009) for the rate-limiting step of this enzyme from the application of the Eyring equation, , to the experimentally observed reaction rate (Begley, Walts & Walsh, 1986b). Since methanethiol is weakly acidic (pKa = 10.4) and is almost completely protonated at physiological pH, the initial cost of deprotonating it must be added to the computations above. This cost amounts to −RT ln 10pH-pKa (Ullmann & Knapp, 1999), or 4.6 kcal mol−1 at pH = 7.

Bottom Line: Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+).Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction.Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

View Article: PubMed Central - HTML - PubMed

Affiliation: FP-ENAS/Fac. de Ciências da Saúde, Universidade Fernando Pessoa , Porto , Portugal.

ABSTRACT
Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

No MeSH data available.