Phosphodiester hydrolysis computed for cluster models of enzymatic active sites

Batebi, Hossein; Imhof, Petra

doi:10.1007/s00214-016-2020-8

Cited by 7 publications

(12 citation statements)

References 78 publications

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“…Asn-212 and Tyr-171 also play key roles at the start of the reaction by positioning the Asp-210 base and the nucleophilic water molecule in the active site, respectively. Although it was previously suggested , that His-309 should be neutral and initiate the reaction by activating a water nucleophile, calculations demonstrate that His-309 must be protonated to help neutralize the charge on the phosphorane intermediate, which is in good agreement with nuclear magnetic resonance and crystallographic data. ,, …”

Section: Introductionsupporting

confidence: 76%

Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis

et al. 2019

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In the repair of the damage to bases, human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key participant via the DNA base excision repair pathway. APE1 cleaves AP sites in DNA, which are potentially cytotoxic and highly mutagenic if left unrepaired. According to existing structural data, this enzyme's active site contains many polar amino acid residues, which form extensive contacts with a DNA substrate. A few alternative catalytic mechanisms of the phosphodiester bond hydrolysis by APE1 have been reported. Here, the kinetics of conformational changes of the enzyme and of DNA substrate molecules were studied during the recognition and cleavage of the abasic site in the pH range from 5.5 to 9.0 using stopped-flow fluorescence techniques. The activity of APE1 increased with an increase in pH because of acceleration of the rates of catalytic complex formation and of the catalytic reaction. Molecular dynamics simulation uncovered a significant increase in the pK a of His-309 located in the active site of the enzyme. This finding revealed that the observed enhancement of enzymatic activity with pH could be associated with deprotonation of not only Tyr-171 but also His-309. The obtained data allowed us to hypothesize that the ionized state of these residues could be a molecular switch between the alternative catalytic mechanisms, which involve different functionalities of these residues throughout the reaction.

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Section: Introductionsupporting

confidence: 76%

Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis

et al. 2019

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“…Asn212 and Tyr171 also play key roles at the start of the reaction by positioning the Asp210 base and the nucleophilic water molecule in the active site, respectively. Although it has been suggested [ 37 , 38 ] that His309 is neutral and initiates the reaction by activating a water nucleophile, calculations made in [ 36 ] indicate that His309 must be protonated to help neutralize the charge on the phosphorane intermediate, in good agreement with NMR and crystallographic data [ 24 , 25 , 39 ].…”

Section: Introductionsupporting

confidence: 54%

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes

Davletgildeeva

Кузнецова

Новопашина

et al. 2022

IJMS

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Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

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“…Our simulation on protonated Asp210 does not reveal any notable conformational changes and proton transfer to the O3’ atom is unlikely to occur due to large distance between the O3’ atom and Asp210. On the other hand, the increased activity of the D210H mutant as opposed to the decreased activity in D210N and D210A can be attributed to a neutral histidine that serves as the general base with a proton affinity that is higher than that of an aspartate residue, a mechanism that has been reported for other endonucleases …”

Section: Discussionmentioning

confidence: 63%

“…At the same time a Mg 2+ ‐ion coordinated to the phosphate group of the AP‐site, either at binding site A or D, could stabilize the abasic DNA for direct attack of the hydroxyl ion . According to previous simulations of cluster models, His309 in the neutral Hsd‐form can indeed act as a proton acceptor, in particular if supported by a nearby aspartate residue such as Asp283.…”

Section: Discussionmentioning

confidence: 94%

Role of AP‐endonuclease (Ape1) active site residues in stabilization of the reactant enzyme‐DNA complex

2018

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Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal-dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg -ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction-competent conformation. Active-site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1-DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule.

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Phosphodiester hydrolysis computed for cluster models of enzymatic active sites

Cited by 7 publications

References 78 publications

Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis

Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes

Role of AP‐endonuclease (Ape1) active site residues in stabilization of the reactant enzyme‐DNA complex

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