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Carbinolamine formation and dehydration in a DNA repair enzyme active site.

Dodson ML, Walker RC, Lloyd RS - PLoS ONE (2012)

Bottom Line: We demonstrated feasible pathways involving water, as well as those independent of water participation.Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed.Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics.

View Article: PubMed Central - PubMed

Affiliation: Active Site Dynamics LLC, Houston, Texas, United States of America. mldodson@comcast.net

ABSTRACT
In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.

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PMF determination for carbinolamine formation by direct proton transfer without involvement of an intermediate water.The work vs. CV functions for each of 30 SMD replicates are given in color in Panel A as is their Jarzynski average (solid black line). For clarity only the Jarzynski average, the bias corrected Jarzynski average  the square root of MSE, and CV definition are shown in Panel B, and is similar to panel B in Fig. 3. As in that figure, the steered direction for carbinolamine formation is right to left.
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pone-0031377-g005: PMF determination for carbinolamine formation by direct proton transfer without involvement of an intermediate water.The work vs. CV functions for each of 30 SMD replicates are given in color in Panel A as is their Jarzynski average (solid black line). For clarity only the Jarzynski average, the bias corrected Jarzynski average the square root of MSE, and CV definition are shown in Panel B, and is similar to panel B in Fig. 3. As in that figure, the steered direction for carbinolamine formation is right to left.

Mentions: Fig. 4 is a stereogram illustrating a typical initial active site quantum region for the SMD simulations further analyzed in Fig. 5 (imine formation without involvement of an intermediate water). The atom names referred to in the CV definition in Fig. 5, Panel B are identified. Interatomic distances are defined similarly to those in Fig. 2.


Carbinolamine formation and dehydration in a DNA repair enzyme active site.

Dodson ML, Walker RC, Lloyd RS - PLoS ONE (2012)

PMF determination for carbinolamine formation by direct proton transfer without involvement of an intermediate water.The work vs. CV functions for each of 30 SMD replicates are given in color in Panel A as is their Jarzynski average (solid black line). For clarity only the Jarzynski average, the bias corrected Jarzynski average  the square root of MSE, and CV definition are shown in Panel B, and is similar to panel B in Fig. 3. As in that figure, the steered direction for carbinolamine formation is right to left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285167&req=5

pone-0031377-g005: PMF determination for carbinolamine formation by direct proton transfer without involvement of an intermediate water.The work vs. CV functions for each of 30 SMD replicates are given in color in Panel A as is their Jarzynski average (solid black line). For clarity only the Jarzynski average, the bias corrected Jarzynski average the square root of MSE, and CV definition are shown in Panel B, and is similar to panel B in Fig. 3. As in that figure, the steered direction for carbinolamine formation is right to left.
Mentions: Fig. 4 is a stereogram illustrating a typical initial active site quantum region for the SMD simulations further analyzed in Fig. 5 (imine formation without involvement of an intermediate water). The atom names referred to in the CV definition in Fig. 5, Panel B are identified. Interatomic distances are defined similarly to those in Fig. 2.

Bottom Line: We demonstrated feasible pathways involving water, as well as those independent of water participation.Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed.Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics.

View Article: PubMed Central - PubMed

Affiliation: Active Site Dynamics LLC, Houston, Texas, United States of America. mldodson@comcast.net

ABSTRACT
In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.

Show MeSH
Related in: MedlinePlus