| 1. |
- Dourado, Daniel F A R, et al.
(författare)
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Glutathione transferase : new model for glutathione activation
- 2008
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 14:31, s. 9591-8
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferases are enzymes of the cellular detoxification system that metabolize a vast spectrum of xenobiotic and endobiotic toxic compounds. They are homodimers or heterodimers and each monomer has an active center composed of a G-site in which glutathione (GSH) binds and an H-site for the electrophilic substrate. When GSH binds to the G-site, the pKa value of its thiol group drops by 2.5 units; this promotes its deprotonation and, therefore, produces a strong nucleophilic thiolate that is able to react with the electrophilic substrate. The mechanism behind the deprotonation of the thiol group is still unknown. Some studies point to the fact that the GSH glutamyl alpha-carboxylate group is essential for GSH activation, whereas others indicate the importance of the active-center water molecules. On the basis of QM/MM calculations, we propose a mechanism of GSH activation in which a water molecule, acting as a bridge, is able to assist in the transfer of the proton from the GSH thiol group to the GSH glutamyl alpha-carboxylate group, after an initial GSH conformational rearrangement. We calculated the potential of mean force of this GSH structural rearrangement that would be necessary for the approach of both groups and we then performed a QM/MM ONIOM scan of water-assisted proton transfer. The overall free-energy barrier for the process is consistent with experimental studies of the enzyme kinetics.
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| 2. |
- Dourado, Daniel F A R, et al.
(författare)
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Glutathione Transferase A1-1 : Catalytic Importance of Arginine 15
- 2010
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:4, s. 1690-1697
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferases (GSTs) are fundamental enzymes of the cell detoxification system. They catalyze the nucleophilic attack Of glutathione (GSH) on electrophilic substrates to produce less toxic compounds. The resulting Substrate can then be recognized by ATP-dependent transmembrane PUMPS and consequently expelled from the cell. Despite all the existing studies on GSTs, many aspects of the catalytic events are still poorly understood. Recently, using as a model the GSTAI-1 enzyme, we proposed it GSH activation mechanism. Resorting to the density functional theory (DFT), we demonstrated that a water molecule could assist a proton transfer between (lie GSH thiol and (x-carboxylic groups. after all initial conformational rearrangement of GSH, as evidenced by potential of mean force calculations. In this work to elucidate the catalytic role of Arg 15, a strictly conserved active site residue in class alpha GSTs. we analyzed the activation energy barrier and Structural details associated with the GSTAI-1 Mutants R15A, R15R epsilon, eta-c (an Arg residue with the epsilon-eta-nitrogens Substituted by carbons), and R 15Rneutral (a neutral Arg residue due to the a addition of a hydride in the zeta-carbon. A similar mechanism to the one used in Our GSH activation proposal was implemented.
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| 3. |
- Dourado, Daniel F. A. R., et al.
(författare)
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Isomerization of Delta(5)-Androstene-3,17-dione into Delta(4)-Androstene-3, 17-dione Catalyzed by Human Glutathione Transferase A3-3 : A Computational Study Identifies a Dual Role for Glutathione
- 2014
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Ingår i: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 118:31, s. 5790-5800
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Delta 5-androstene-3,17-dione (Delta(5)-AD) and Delta(5)-pregnene-3,20-dione (Delta(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Delta(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Delta(5)-AD C4 atom to the Delta(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal.mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the 03 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leul11, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Delta(4)-AD crystal structure and is consistent with all available experimental data.
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| 4. |
- Dourado, Daniel F. A. R., et al.
(författare)
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Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:45, s. 8069-8078
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Tidskriftsartikel (refereegranskat)abstract
- Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.
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| 5. |
- Zhang, Wei, et al.
(författare)
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Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates
- 2015
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006. ; 1850:4, s. 742-749
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Tidskriftsartikel (refereegranskat)abstract
- Background: Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective. Methods: In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity. Results: Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis. Conclusions: We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs. General significance: The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.
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| 6. |
- Zhang, Wei, et al.
(författare)
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Multidimensional epistasis and fitness landscapes in enzyme evolution
- 2012
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 445, s. 39-46
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Tidskriftsartikel (refereegranskat)abstract
- The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the imitations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.
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