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ABSTRACT: Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide substrates. In the present study, combined quantum mechanical/molecular mechanical (QM/MM) simulations of the conjugation of glutathione to phenanthrene 9,10-oxide, catalyzed by the M1-1 isoenzyme from rat, have been carried out to obtain insight into details of the reaction mechanism and the role of solvent present in the highly solvent accessible active site. Reaction-specific AM1 parameters for sulfur have been developed to obtain an accurate modeling of the reaction, and QM/MM solvent interactions in the model have been calibrated. Free energy profiles for the formation of two diastereomeric products were obtained from molecular dynamics simulations of the enzyme, using umbrella sampling and weighted histogram analysis techniques. The barriers (20 kcal/mol) are in good agreement with the overall experimental rate constant and with the formation of equal amounts of the two diastereomeric products, as experimentally observed. Along the reaction pathway, desolvation of the thiolate sulfur of glutathione is observed, in agreement with solvent isotope experiments, as well as increased solvation of the epoxide oxygen of phenanthrene 9,10-oxide, illustrating an important stabilizing role for active site solvent molecules. Important active site interactions have been identified and analyzed. The catalytic effect of Tyr115 through a direct hydrogen bond with the epoxide oxygen of the substrate, which was proposed on the basis of the crystal structure of the (9S,10S) product complex, is supported by the simulations. The indirect interaction through a mediating water molecule, observed in the crystal structure of the (9R,10R) product complex, cannot be confirmed to play a role in the conjugation step. A selection of mutations is modeled. The Asn8Asp mutation, representing one of the differences between the M1-1 and M2-2 isoenzymes, is identified as a possible factor contributing to the difference in the ratio of product formation by these two isoenzymes. The QM/MM reaction pathway simulations provide new and detailed insight into the reaction mechanism of this important class of detoxifying enzymes and illustrate the potential of QM/MM modeling to complement experimental data on enzyme reaction mechanisms
Journal of the American Chemical Society 124 (2002) 33.
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ABSTRACT: Modeling methods allow the identification and analysis of determinants of reactivity and specificity in enzymes. The reaction between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) is widely used as a standard activity assay for glutathione S-transferases (GSTs). It is important to understand the causes of differences between catalytic GST isoenzymes and the effects of mutations and genetic polymorphisms. Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations have been performed here to investigate the addition of the glutathione anion to CDNB in the wild-type M1-1 GST isoenzyme from rat and in three single point mutant (Tyr6Phe, Tyr115Phe, and Met108Ala) M1-1 GST enzymes. We have developed a specifically parameterized QM/MM method (AM1-SRP/CHARMM22) to model this reaction by fitting to experimental heats of formation and ionization potentials. Free energy profiles were obtained from molecular dynamics simulations of the reaction using umbrella sampling and weighted histogram analysis techniques. The reaction in solution has also been simulated and is compared to the enzymatic reaction. The free energies are in excellent agreement with experimental results. Overall the results of the present study show that QM/MM reaction pathway analysis provides detailed insight into the chemistry of GST and can be used to obtain mechanistic insight into the effects of specific mutations on this catalytic process.
Biochemistry 46 (2007) 21.
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The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 107 (2003).
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ABSTRACT: A combined quantum mechanical and molecular mechanical (QM/MM) method (AM1/CHARMM) was used to investigate the mechanism of the aromatic hydroxylation of phenol by a flavin dependent phenol hydroxylase (PH), an essential reaction in the degradation of a wide range of aromatic compounds. The model for the reactive flavin intermediate (C4a-hydroperoxyflavin) bound to PH was constructed on the basis of the crystal structure of the enzyme-substrate complex. A potential energy surface (PES) was calculated as a function of the reaction coordinates for hydroxylation of phenol (on C6) and for proton transfer from phenol (O1) to an active-site base Asp54 (OD1). The results support a reaction mechanism in which phenol is activated through deprotonation by Asp54, after which the phenolate is hydroxylated through an electrophilic aromatic substitution. Ab initio test calculations were performed to verify these results of the QM/MM model. Furthermore, the variation in the calculated QM/MM activation energies for hydroxylation of a series of substrate derivatives was shown to correlate very well (R = 0.98) with the natural logarithm of the experimental rate constants for their overall conversion by PH (25 C, pH 7.6). This correlation validates the present QM/MM model and supports the proposal of an electrophilic aromatic substitution mechanism in which the electrophilic attack of the C4a-hydroperoxyflavin cofactor on the activated (deprotonated) substrate is the rate-limiting step at 25 C and pH 7.6. The correlation demonstrates the potential of the QM/MM technique for predictions of catalytic activity on the basis of protein structure. Analysis of the residue contributions identifies a catalytic role for the backbone carbonyl of a conserved proline residue, Pro364, in specific stabilization of the transition state for hydroxylation. A crystal water appears to assist in the hydroxylation reaction by stabilizing the deprotonated C4a-hydroxyflavin product. Comparison of the present results with previous QM/MM results for the related p-hydroxybenzoate hydroxylase (Ridder et al. J. Am. Chem. Soc. 1998, 120, 7641-7642) identifies common mechanistic features, providing detailed insight into the relationship between these enzymes.
Journal of the American Chemical Society 122 (2000).
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Journal of the American Chemical Society 124 (2002).
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J. Am. Chem. Soc. 120 (1998) 7641-7642.
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ABSTRACT: The reaction pathway for the aromatic 3-hydroxylation of p-hydroxybenzoate by the reactive C4a-hydroperoxyflavin cofactor intermediate in p-hydroxybenzoate hydroxylase (PHBH) has been investigated by a combined quantum mechanical and molecular mechanical (QM/MM) method. A structural model for the C4a-hydroperoxyflavin intermediate in the PHBH reaction cycle was built on the basis of the crystal structure coordinates of the enzyme-substrate complex. A reaction pathway for the subsequent hydroxylation step was calculated by imposing a reaction coordinate that involves cleavage of the peroxide oxygen-oxygen bond and formation of the carbon-oxygen bond between the C3 atom of the substrate and the distal oxygen of the peroxide moiety of the cofactor. The geometric changes and the Mulliken charge distributions along the calculated reaction pathway are in line with an electrophilic aromatic substitution type of mechanism. The energy barrier of the calculated reaction is considerably lower when the substrate hydroxyl moiety is deprotonated, in comparison with the barrier found with a protonated hydroxyl moiety. This effect of the protonation state of the substrate on the calculated energy barrier supports experimental observations that deprotonation is required for hydroxylation of the substrate. A notable event in the calculated reaction pathway is a lengthening of the peroxide oxygen-oxygen bond at an intermediate stage. Further analysis of the reaction pathway indicates that this oxygen-oxygen bond elongation is accompanied by an increase in electrophilic reactivity on the distal oxygen of the peroxide moiety, which may assist the C-O bond formation in the reaction of the C4a-hydroperoxyflavin intermediate with the substrate. Analysis of the effect of individual active site residues on the reaction reveals a specific transition state stabilization by the backbone carbonyl moiety of Pro293. The crystal water 717 appears to drive the hydroxylation step through a stabilizing hydrogen bond interaction to the proximal oxygen of the C4a-hydroperoxyflavin intermediate, which increases in strength as the hydroperoxyflavin cofactor converts to the anionic (deprotonated) hydroxyflavin.
Journal of Molecular Graphics and Modelling 17(3-4):163-75, 214. · 2.18 Impact Factor
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Chemical Communications.
