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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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Reactions catalyzed by lyase-capable BER glycosylases.This diagram illustrates the possible reaction pathways catalyzed by these enzymes. The proportions of abasic sites and – and –elimination products in the product spectra depend on the intrinsic chemistry of the imine intermediates and the relative rates of their (1) hydrolytic turnover, e.g., Schiff base intermediateE–Abasic site and (2) conversion to “downstream” products, e.g., Schiff base intermediateE–SSB (covalent). This report mainly investigates the reactions corresponding to E–Abasic site intermediateSchiff base in the figure: the collapse of the amine nitrogen onto the carbonyl carbon to form the carbinolamine intermediate followed by its dehydration. These reactions are outlined by the rectangular box in the figure. E–DNA may represent the enzyme bound at the location of either an abasic site, a pyrimidine photodimer or a lyase product of the – or –elimination type.
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pone-0031377-g001: Reactions catalyzed by lyase-capable BER glycosylases.This diagram illustrates the possible reaction pathways catalyzed by these enzymes. The proportions of abasic sites and – and –elimination products in the product spectra depend on the intrinsic chemistry of the imine intermediates and the relative rates of their (1) hydrolytic turnover, e.g., Schiff base intermediateE–Abasic site and (2) conversion to “downstream” products, e.g., Schiff base intermediateE–SSB (covalent). This report mainly investigates the reactions corresponding to E–Abasic site intermediateSchiff base in the figure: the collapse of the amine nitrogen onto the carbonyl carbon to form the carbinolamine intermediate followed by its dehydration. These reactions are outlined by the rectangular box in the figure. E–DNA may represent the enzyme bound at the location of either an abasic site, a pyrimidine photodimer or a lyase product of the – or –elimination type.

Mentions: The various BER enzymes fall into two broad categories based on mechanism: those in which reactions subsequent to glycosyl bond scission may involve lyase–type DNA sugar–phosphate backbone cleavages (–elimination and, possibly, –elimination), and those which leave the DNA backbone intact, e.g., uracil DNA glycosylase. T4PDG initiates repair at pyrimidine ultraviolet photodimers [2], [3] and furnishes an example of the steps, subsequent to glycosyl bond cleavage, characteristic of the first kind of BER enzyme (Fig. 1). The lyase steps, – and –elimination, proceed via DNA deoxyribose –imine (Schiff base) intermediates in which the amine is the N–terminal threonine –amine. The protonated imine is thought to act as an electrophilic catalyst [4] for the sequential – and –elimination reactions [2], [3], [5].


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

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

Reactions catalyzed by lyase-capable BER glycosylases.This diagram illustrates the possible reaction pathways catalyzed by these enzymes. The proportions of abasic sites and – and –elimination products in the product spectra depend on the intrinsic chemistry of the imine intermediates and the relative rates of their (1) hydrolytic turnover, e.g., Schiff base intermediateE–Abasic site and (2) conversion to “downstream” products, e.g., Schiff base intermediateE–SSB (covalent). This report mainly investigates the reactions corresponding to E–Abasic site intermediateSchiff base in the figure: the collapse of the amine nitrogen onto the carbonyl carbon to form the carbinolamine intermediate followed by its dehydration. These reactions are outlined by the rectangular box in the figure. E–DNA may represent the enzyme bound at the location of either an abasic site, a pyrimidine photodimer or a lyase product of the – or –elimination type.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0031377-g001: Reactions catalyzed by lyase-capable BER glycosylases.This diagram illustrates the possible reaction pathways catalyzed by these enzymes. The proportions of abasic sites and – and –elimination products in the product spectra depend on the intrinsic chemistry of the imine intermediates and the relative rates of their (1) hydrolytic turnover, e.g., Schiff base intermediateE–Abasic site and (2) conversion to “downstream” products, e.g., Schiff base intermediateE–SSB (covalent). This report mainly investigates the reactions corresponding to E–Abasic site intermediateSchiff base in the figure: the collapse of the amine nitrogen onto the carbonyl carbon to form the carbinolamine intermediate followed by its dehydration. These reactions are outlined by the rectangular box in the figure. E–DNA may represent the enzyme bound at the location of either an abasic site, a pyrimidine photodimer or a lyase product of the – or –elimination type.
Mentions: The various BER enzymes fall into two broad categories based on mechanism: those in which reactions subsequent to glycosyl bond scission may involve lyase–type DNA sugar–phosphate backbone cleavages (–elimination and, possibly, –elimination), and those which leave the DNA backbone intact, e.g., uracil DNA glycosylase. T4PDG initiates repair at pyrimidine ultraviolet photodimers [2], [3] and furnishes an example of the steps, subsequent to glycosyl bond cleavage, characteristic of the first kind of BER enzyme (Fig. 1). The lyase steps, – and –elimination, proceed via DNA deoxyribose –imine (Schiff base) intermediates in which the amine is the N–terminal threonine –amine. The protonated imine is thought to act as an electrophilic catalyst [4] for the sequential – and –elimination reactions [2], [3], [5].

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