This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In an extension of our work on Aldehyde Dehydrogenase (see: Section II.C.1) and to establish general principles describing cysteine nucleophilic attack on electrophilic substrates in enzymatic systems, we have investigated the detailed binding mechanism of an aromatic aldehyde to a Glutathione S-Transferase (GST). Analysis of MD and QM/MM simulations suggests that interactions of a proximal residue with an active site residue facilitates the approach and binding of the substrate in GSTs. Once a reaction between a substrate and human pi-class Glutathione S-Transferase (GSTP1-1)/glutathione is complete, the GSH-substrate conjugate can disassociate from the enzyme and hence the usually toxic substrate is more readily excreted from the body. Therefore, understanding GST activity provides crucial insight into the human health implications of certain toxins. The lowering of the cysteine pKa within GSH upon binding to GST (~9 in solution versus 6-7 once bound) is well established [1]. This change in pKa reveals that this cysteine predominantly exists in its negatively charged state (a thiolate anion, GS-). This thiolate anion can be stabilized by interaction with the hydroxyl group of Tyr7. Whether this tyrosine can play the role of general base or the proton is equally shared between these two residues has been the subject of some controversy [2]. We performed molecular dynamics (MD) simulations of water solvated GSTP1-1 in its holo form using CHARMM and the all22 molecular mechanics (MM) force field. This simulation was performed using Configuration 1 which has been shown to be the major population in holo form GSTP1-1. In the substrate bound form, we considered the hypothesis that the hydroxyl proton on Tyr7 can be transferred to Cys-GS-. Therefore, we simulated both configurations to determine which binds the benzaldehyde substrate in conformations conducive to reaction. Analysis of the results in terms of near-attack conformers (NACs) produced from these simulations reveals that in Configuration 1 the enzyme/cofactor system is much less effective at binding substrate than when in Configuration 2. The percentage of NACs is 0.0 % (Configuration 1) and 4.9 % (Configuration 2) with a major fraction just outside the NAC region. Furthermore, the conformations closest to the NAC region occur only during the initial part while in Configuration 1 system for which the system may not have equilibrated while in Configuration 2 the substrate actually exits the binding site and then enters again forming more NACs. This result is similar to that observed in the MD simulations of benzaldehyde in the active site of Aldehyde Dehydrogenase. Thiolate anions have very high solvation energy [3], thus replacing the interactions of water molecules with those of the substrate will be energetically unfavorable and thus the substrate seems to prefer to bind to a protonated cysteine. Finally, we have calculated the free energy profile for transferring the shared proton between Cys-GS- and Tyr-7. For these simulations we used a hybrid Quantum Mechanical (QM)/MM potential function present in the DYNAMO program. An appropriate reaction coordinate, q, for the proton transfer between these two residues is: q= [1/(ms + mo)](ms rSH - mo rOH) where rSH and rOH are the distances of the proton from the donor sulfur and the acceptor oxygen respectively with ms and mo as their masses. The PM3 potential function (used as the QM method in the QM/MM simulations) most likely underestimates the stability of the tyrosinate anion and slightly overestimates the stability of a configuration where the proton is located almost equidistant between both residues yet the relative energetics of this proton transfer are qualitatively in agreement with high-level ab inito (MP2) calculations. We also examined another QM semiempirical method, AM1, and discovered that it is totally inappropriate for studies of this proton transfer. The free energy QM/MM simulations show the proton is energetically favored to be located on Tyr7 in the holo form GST, in accordance with experiment. Yet in the substrate bound form, the proton prefers to be located closer to Cys-GS-. Substrate binding lowers the free energy difference between these two states by 6 kcal/mol. The results of MM simulations and QM/MM simulations suggest that upon approach of a substrate into the active site of GST, the proton on Tyr7 can shift to be located closer to the Cys-GS- enabling it to bind substrate. With the general details of the mechanism becoming clearer, we will begin examining the role of residues proximal to the active site in defining substrate specificity. As in the case of the ALDH work, we will use the 'Group Entropy' (GEnt) analysis to assist in the identification of residues for further additional analysis. Our goal is to begin defining a model for the GSTs that provides insights into its biochemical and physiological roles. The computations were performed on the Terascale Computing System at the PSC. References: 1.Graminski, G. F., Kubo, Y., Armstrong, R. N. 1989. Biochemistry, 28:3562-3568. 2.Kolm, R. H., Sroga, G., Mannervik, B. 1992. Biochem. J., 285:537-540. 3.Pliego, J. R. and Riveros, J. M. 2002. Phys. Chem. Chem. Phys., 4:1622-1627. This material was presented at the 227th National Meeting of the American Chemical Society on March 28-April 1, 2004 in Anaheim, CA.
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