A Comprehensive Understanding of Enzymatic Degradation of the G

Research Article Cite This: ACS Catal. 2019, 9, 7038−7051

pubs.acs.org/acscatalysis

A Comprehensive Understanding of Enzymatic Degradation of the G‑Type Nerve Agent by Phosphotriesterase: Revised Role of Water Molecules and Rate-Limiting Product Release Fangfang Fan,†,§ Yongchao Zheng,‡,§ Yuwei Zhang,† He Zheng,‡ Jinyi Zhong,*,‡ and Zexing Cao*,†

Downloaded via BUFFALO STATE on July 31, 2019 at 15:57:17 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 360015, People’s Republic of China ‡ State Key Laboratory of NBC Protection for Civilian, Beijing 102205, People’s Republic of China S Supporting Information *

ABSTRACT: Nerve agents are highly toxic organophosphorus compounds, and the wild-type phosphotriesterase (PTE) enzyme is capable of hydrolyzing these organophosphates but with a low catalytic efficiency. Here the whole enzymatic detoxification process of the G-type nerve agent sarin by the PTE enzyme, including the substrate delivery, the chemical reaction, and the product release, has been explored by extensive QM/MM MD and MM MD simulations. The plausible mechanisms for the chemical and nonchemical steps, the roles of water molecules, and the key residues have been discussed. The enzymatic P−F cleavage of sarin is a two-step exothermic process with the free-energy span of 12.3 kcal/ mol, and it should be facile in the whole enzymatic catalysis. On the contrary, the initial degraded product is tightly bound to the binuclear zinc center, and its dissociation experiences multiple chemical steps with the free-energy barriers of 21.0 kcal/mol for the recombination process and 18.3 kcal/mol for the release of the product phosphoester from the active site. Notably, the solvation of hydrophilic products in the bulk water is generally exothermic, which provides the driving force for the release of products from the active site. The side-chain residues Leu271 and Phe132 in the transportation channel function as the entrance gate in PTE and play an important gate-switching role to manipulate the substrate access to the active site and the product release. These mechanistic details for the enzymatic degradation of sarin by PTE provide significant clues to improve its activity toward the nerve agents. KEYWORDS: enzymatic detoxification, nerve agent, sarin, phosphotriesterase, catalytic mechanism, QM/MM MD and MM MD simulations

1. INTRODUCTION Phosphotriesterase (PTE), as a member of the amidohydrolase superfamily, catalyzes the detoxification of organophosphorus compounds chiefly used as pesticides and nerve agents. The high-resolution X-ray crystal structure of PTE reveals a subunit of homodimeric (β/α)8-barrel, in which the active site is surrounded by the eight loops bridging the core β-strands and the subsequent α-helices (Figure 1).1 These loops may remarkably influence the enzymatic activity, and in particular, the largest loop 7 is responsible for the substrate binding and delivery.2,3 As Figure 1A shows, the active domain has three pockets, named as large pocket, small pocket, and leaving pocket. The small pocket is relatively isolated from the external environment, and the large pocket is accessible to solvent molecules.4 Both small and large pockets accommodate the substituent group of the ligand and control the stereochemical selectivity of PTE. The leaving pocket is exposed to the solvent, which plays a crucial role in coordinating delivery of the © XXXX American Chemical Society

substrate. This kind of enzyme usually utilizes one or two divalent metal ions to activate a hydroxide ion for the nucleophilic attack at the phosphorus center in hydrolysis. Here the active site in the wild-type PTE enzyme is located at the C-terminal end of the barrel and contains two zinc ions coordinated by four histidine residues (H55, H57, H201 and H230), an aspartic acid (D301), a carboxylated lysine (K169), and a hydroxide ion (Figure 1B).1,5 Organophosphates (OPs) are generally of high toxicity, and they have been widely used as insecticides and chemical nerve agents, in which the central phosphorus atom, characterized by a PO or PS double bond, is bonded to alkoxy or amino groups and to another halogen, aliphatic, or aromatic moiety as the leaving group (see Scheme 1).6 The organophosphate Received: May 7, 2019 Revised: June 24, 2019 Published: July 1, 2019 7038

DOI: 10.1021/acscatal.9b01877 ACS Catal. 2019, 9, 7038−7051

Research Article

ACS Catalysis

Figure 1. (A) Three pockets of the substrate-binding region: large pocket, small pocket, and leaving pocket. (B) (β/α)8-barrel structure of PTE, eight loops bridging the core β-strands and α-helices, and the detailed coordination of the binuclear zinc center highlighted in the dotted box.

site.14−16 AChE catalyzes the hydrolytic breakdown of the neurotransmitter acetylcholine, which has a function in different nervous systems of vertebrates.17−19 The overaccumulation of acetylcholine at neuron−neuron junctions will result in failure of many physiological functions, even death,20−22 and thus the toxicity of OPs can be measured by targeting AChE. In dealing with nerve agents, there has been a long-standing interest in enzymatic detoxification/decontamination because of its environmental friendliness compared with the conventional chemical treatment and incineration.23 Currently, organophosphate-degrading enzymes, such as the wild-type and variant forms of PTE, are promising candidates and have been used for the degradation of organophosphate com-

pesticides have been widely used to control weeds and pests for the crop production in modern agriculture. However, these OPs are highly stable on the earth, and they have a half-life over millions of years during the hydrolytic degradation in water.7,8 Accordingly, the heavy and widespread use of pesticides will cause serious environmental and human health issues. The extremely toxic organophosphate compounds, such as the G-type nerve agent sarin and the V-type nerve agent VX, have been employed as chemical weapons in terrorist attacks and wars,9,10 and their long-term effects are still not well understood at present.11−13 The toxicity of nerve agents is rooted in the irreversible inhibition of acetylcholinesterase (AChE) through the covalent phosphorylation of the serine residue in the active 7039


Research Article

ACS Catalysis Scheme 1. Selected Organophosphates Used As Pesticides and Nerve Agents as well as the Enzymatic Hydrolysis

Figure 2. Reaction coordinate (RC1) defined by the distance between the O1 of sarin and the CA of Glu56 for the substrate delivery to the active site.

pounds.24,25 In particular, PTE shows wide substrate specificity, but the hydrolysis rates strongly depend on the chemical features of organophosphate substrates. For example, the best known substrate for PTE is the insecticide paraoxon with an high enzymatic efficiency (kcat/Km ≈ 108 M−1 s−1), which is comparable with the diffusion-dominated limit.26−29 However, for hydrolysis of the G-type nerve agents by PTE, the catalytic efficiency is reduced to 104 ∼ 105 M−1 s−1, and even much lower for the V-type nerve agents.30,31 In order to understand this enzymatic process and improve the activity of PTE toward the detoxification of nerve agents, many efforts have been made in the past decades.32−35 However, because of the high toxicity of nerve agents and the expensive costs for the experimental evaluation of the enzymatic activity, few analogue substrates have been explored experimentally up to now.36,37 Curiously, the computational work on this subject remains limited,26,38,39 and the detailed mechanisms for the detoxification process are still less known. It is well-known that the entire enzymatic process generally includes the substrate transportation and binding to the active site, the catalytic reaction, and the product release, in which both chemical and nonchemical steps are significant for the enzymatic efficiency.40 Here extensive QM/MM and MM MD simulations have been carried out. We intend to build a full picture of the catalytic detoxification of the nerve agent sarin by PTE, and thus, the following key issues will be addressed in the present study: (i) the dynamic and thermodynamic features for the substrate delivery, the catalytic reaction, and the release of products in the enzymatic hydrolysis of sarin by PTE; (ii) the plausible channels for the delivery of substrate and product; (iii) the practical structure of the active site and the rate-limiting step; and (iv) the role of two Zn2+, key residues, and water molecules in the catalytic reaction and the substrate/product transportation. A comprehensive understanding of the whole enzymatic catalysis is highly required for the development of a sustainable means to deal with the nerve agent.

corresponding 3.5 Å observed in the diethyl 4-methylbenzyl phosphonate-enzyme complex (PDB ID: 1DPM).41 Here both A and B chains of the enzyme were considered in the computational model, in which the inhibitor was modified into the sarin substrate. The protonation states of all ionizable residues were assigned by using the PROPKA42 package at pH 7.0 according to the experimental condition. The protonation state of each histidine residue was determined by considering the possible hydrogen-bonding network with its neighboring groups in the crystal structure. The substrate and protein are described by the AMBER GAFF force field43 and the AMBER14SB force field,44 respectively. The partial atomic charges of the substrate were assigned by the restrained electrostatic potential (RESP) charge45,46 by using the HF/631G* calculation with the Gaussian 0947 package. The whole system was solvated into a rectangular TIP3P48 water box of ∼98 Å × 108 Å × 105 Å, and two chloride counterions were added to neutralize the model system. The initial coordinates and topology parameters were generated by the tleap tool in AMBER 18.49 Classical MD Simulations. All model systems were optimized by the three-step energy minimizations at the MM level to adjust poor interatomic interactions. First, the water molecules were minimized while keeping the protein and substrate constrained. Then, the side chains were allowed to relax while the main chains of protein are restrained. Finally, the entire system was completely relaxed without any restriction. For each minimization step, the conjugate gradient iterations were carried out for 5000 cycles after performing 5000 step steepest descent energy minimization. The optimized system was heated up from 0 to 300 K gradually under the NVT ensemble for 100 ps, followed by 100 ps of equilibration under the NPT ensemble to relax the system density to about 1.0 g/ cm3. Finally, a 200 ns MD simulation under the NPT ensemble was carried out with an integration time step of 1 fs based on the periodic boundary condition. In the long-time MD simulations, the SHAKE algorithm50 was applied to constrain all hydrogencontaining bonds with a tolerance of 10−5, and a 12 Å cutoff was set for both van der Waals and electrostatic interactions. For these models considered here, the last 150 ns MD simulation indicates that the equilibration of system may be reached, based on the root-mean-square deviation (RMSD) values. The final snapshot from the stable trajectories was used to prepare the

2. COMPUTATIONAL METHODS Setup of the Enzyme−Substrate Complex Model. The initial PTE-substrate model was prepared on the basis of the Xray crystal structure of phosphotriesterase (PTE) from Pseudomonas diminuta complexed with an inhibitor of the diisopropyl methyl phosphonate compound, which serves as an analogue for sarin (PDB ID: 1EZ2).1 The distance between the phosphoryl and Znβ is 2.5 Å, much shorter than the 7040


Research Article

ACS Catalysis

Figure 3. Possible channels of the substrate delivery to the active pocket.

center of mass for the ligand to identify the possible pathways for the ligand fleeing away from the protein within a certain period of time. If the ligand displacement could not meet the threshold parameter, a new random direction will be selected; otherwise, the direction was maintained. Upon the escape of ligand from the initial position, the conventional MD simulations would be switched on and the equilibration sampling was recovered. Herein, the random accelerations of 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, and 0.60 kcal Å−1 g−1 were applied to the initial structure and all of these combinations above were repeated 10 times. Finally, 70 RAMD-MD trajectories for each model were identified. According to the trajectory statistics of RAMD-MD simulations, the possible channels can be determined. Along the most probable channel determined from RAMD-MD simulations, the distance between the O1 of sarin and the CA of Glu56 was chosen as the reaction coordinate (RC1) for the substrate transportation (see Figure 2), which varies from 20.6 to 9.6 Å with a 0.4 Å interval for two adjacent windows, and 28 windows were selected. For each window, 13 ns MD simulations with the appropriate biasing harmonic potential (10−55 kcal/ mol) along the path of the substrate delivery were carried out. The last 5 ns reaction coordinate data for all windows were analyzed by the weighted histogram analysis method (WHAM) to generate the potential of mean force (PMF).

QM/MM model. All classical MD simulations were accomplished by using the AMBER 18 software.49 QM/MM MD Simulations. QM/MM models for the reaction step were prepared on the basis of the equilibrated enzyme−substrate system from the classical MD simulations. The resulting QM/MM system consists of ∼16 000 atoms. The QM subsystem consists of more than 100 atoms, including His55, His57, Lys169, His201, His230, Asp233, His 254, Asp301, two zinc ions, substrate, and water molecules in the active site, which were directly involved in the chemical reaction and the release of products. The QM region was treated by using the B3LYP functional and the def2-SVP and def2/J auxiliary basis sets. The rest of the protein and solvent is described by using the atomic force fields at the MM level, which was the same as that used in the above classical MD simulations. The spherical boundary condition was applied, in which the atoms more than 20 Å away from the spherical center were fixed. The 18 and 12 Å cutoffs were employed for the electrostatic and van der Waals interactions, respectively. In the QM/MM treatment for the chemical reaction steps, different reaction coordinates were defined by the combination of related interatomic distances. The minimum energy path (MEP) for the catalytic step was mapped out by the reaction coordinate driving method, on the basis of the QM/MM minimization calculation. Then, a 500 ps MD simulation was performed to equilibrate the MM part with the QM subsystem constrained. Finally, the ab initio QM/MM MD simulations combined with the umbrella sampling were carried out. Each window was simulated for 20 ps with the time step of 1 fs for the configurational sampling, and the last 10 ps trajectories were collected to determine the free-energy profile. The probability distributions along the reaction coordinate were determined for each window and pieced together with the WHAM technology51 to calculate the potential of mean force (PMF).52,53 All our QM/ MM MD calculations were performed with the modified ORCA 4.054 and AMBER 18 programs.49 RAMD MD Simulations. In order to figure out possible channels for the substrate delivery to the active site, the combined random acceleration molecular dynamics and MD simulations (RAMD-MD) have been carried out by using NAMD 2.9,55 which has been successfully used in previous theoretical studies.56−59 In the RAMD-MD simulations, an additional force with the random orientation was added to the

3. RESULTS AND DISCUSSION 3.1. Possible Delivery Channels for the Nerve Agent Sarin. The possible pathways for the substrate delivery from the outside to the active sites were initially identified by RAMD-MD simulations. As shown in Figure 3, three possible channels, named as Pa, Pb, and Pc, were determined. Path Pa, along the leaving pocket, has a dominant share of trajectories (∼ 72.9%). While the paths Pb and Pc, located among helices 13, 14, 15 and helices 3, 4, 15, respectively, are only respectively responsible for 14 and 5 out of total 70 trajectories. Accordingly, the channel Pa is the most probable pathway for the substrate delivery to the active pocket, and the transportation mechanism as well as the thermodynamic and dynamic properties will be discussed in detail by using classical MD simulations in combination with the umbrella sampling. Here the reaction coordinate RC1 (see Figure 2) was selected to determine the relative free-energy profiles for the substrate delivery. 7041


Research Article

ACS Catalysis

Figure 4. Representative structures and conformational changes of the PTE-substrate system during the sarin transportation to the active site.

(ii) The intermediate stage (16.8 Å > RC1 ≥ 13.6 Å). Guided by the interaction between the phosphoryl and the positive charged zinc ions, the substrate goes deeply into the pocket and approaches the more solvent exposed Znβ gradually, and this kind of interaction provides a driving force to assist the substrate delivery and binding. At RC1 = 14.8 Å, the distance between the phosphoryl and Znβ is reduced to 2.2 Å, and the loop7 partially evolves into a spiral conformation, as shown in Figure 4. We note that the diameter of the entrance gate increases to 18.35 Å and reaches its maximum size during the substrate transportation (see Figure 5), indicating that the substrate passes across the narrowest part of the pocket at this stage. (iii) The final stage (13.6 Å > RC1 ≥ 9.6 Å). Once the substrate passes through the entrance gate successfully, it begins to adjust its posture to adapt to the active site environment. With the advantage of small size and high flexibility, the central phosphorus group enters into the

3.2. Regulation Mechanism of the Substrate Transportation. As Figure 4 shows, the whole transportation process of sarin to the active site can be approximately divided into consecutive three stages: (i) The initial stage (RC1 ≥ 16.8 Å). The ligand is completely free outside the protein when RC1 ≥ 21 Å, and then the substrate approaches the leaving pocket gradually through the interaction with the residues from loop7, giving rise to the destruction of the helix into a flexible loop when RC1 = 20.2 Å. This kind of destruction lasts until the ligand crosses this loop and enters into the pocket at RC1 = 17.4 Å. As the substrate gets into the pocket, the residues Leu271 and Phe132 function as the entrance gate in PTE, as shown by the evolution of distance between Leu271@ CD1 and Phe132@CA in Figure 5. Such gate movement of these residues arises from the interaction between sarin and loop7, and makes the leaving pocket further covered by the loop7. 7042


Research Article

ACS Catalysis

Figure 5. Representative structures and conformational changes of the delivery channel in the sarin transportation.

relevant pocket first, and then the bulky group follows, as shown in Figure 4. At this stage, the substrate sarin is completely immersed in the pocket, and an appropriate binding mode appears at RC = 9.6 Å. As Figure 5 shows, with the substrate moving into the pocket, the door of the delivery channel undergoes “half closed−closed” conformational changes, along with the decrease of the channel diameter characterized by the distance between Leu27 and Phe132. Most notably, the number of water molecules in the pocket experiences two sharp drops at RC = 13.0 and 10.0 Å, i.e., the beginning and ending in this stage, as shown in Figure 6. We note that the number of water molecules around two zinc ions remains dynamically stable in other periods during the substrate transportation. The evolution features of the number of water molecules suggest that the substrate undergoes the posture relaxation in the active site before its binding to

the pocket completely, and quite a few of water molecules are bound in the pocket. These water molecules play an important role in the substrate binding, since the squeezing out of water molecules solvating the protein active site from the pocket may remarkably contribute the binding affinity of the substrate due to the desolvation free energy from their displacement into the bulk solvent. As Figure 7A shows, the substrate transportation from outside to the active site is exothermic by 16.8 kcal/mol. More importantly, the diameter of transportation channel experiences continuous fluctuations as sarin enters into the active site, and the whole delivery process of sarin can be divided into three stages, as shown in Figure 7B. Loop7, served as a door keeper, regulates the contact and interaction of substrate with the entrance gate, and plays a crucial role in the substrate accessing into the active site. 7043


Research Article

ACS Catalysis

Figure 6. Statistics analysis of water molecules around the binuclear zinc ions within 5 and 6 Å, respectively.

Figure 8. Reaction coordinate (RC2) for the initial chemical reaction in the QM/MM calculation.

3.3. Chemical Reaction Mechanisms for the Hydrolysis of Sarin. A key feature of the active site of PTE is a binuclear zinc-center (see Figure 1), in which each zinc ion is coordinated by five residues in the initial crystal structure, and this structural model has been widely used in previous studies.38,26,9,5 During the setup of our computational model, the long-term MD simulations reveal that two water molecule subsequently enter into the binding pocket and are coordinated to two zinc ions respectively. Consequently, two zinc ions become six-coordinated with one stable Zn-bound water molecule during a 200 ns MD simulation, as shown in Figure S1, in which the average Zn···OH2 distances are 1.95 ± 0.07 and 1.98 ± 0.09 Å, respectively. Accordingly, the feasible structure of the binuclear six-coordinated zinc center is used in the present study. In the current model, the substrate sarin binds to the active site by the bonding interactions of its divalent phosphoryl oxygen with the more solvent exposed Znβ. The two zinc ions in the active site are bridged by the carboxylate group (Lys169) and the nucleophile hydroxide ion, and each zinc has a stable hexacoordinate structure with one additional water molecule.

The presence of Zn-bound water molecules has a huge impact on the catalytic activity (vide infra). It is widely accepted that the OP hydrolysis initiates from the nucleophilic attack of the bridging−OH on the phosphorus center, and thus the distance difference of r2−r1 was chosen as the reaction coordinate (RC2) (see Figure 8) to describes the P−F cleavage and P−Oμ formation in the hydrolytic reaction in QM/MM MD simulations. Current QM/MM calculations and QM/MM MD simulations reveal that the initial chemical process follows a two-step stepwise mechanism, including the P−Oμ bond formation arising from the nucleophilic attack of the bridging hydroxide group and the P−F bond cleavage coupled with the hydrogen (Hμ) transfer to the residue Asp301. The selected representative structures, the evolution of selected interatomic distances, and the predicted relative free-energy profiles along the hydrolytic reaction are presented in Figures 9, 10, and 11, respectively. As Figure 9 shows, at the initial state, the Asp301···μ−OH hydrogen-bond interaction is identified, and the distances of

Figure 7. (A) Free-energy profiles for the sarin delivery to the active site. (B) The diameter fluctuation of the transportation channel characterized by the distance between Leu271 and Phe132 in the sarin transportation for three stages of I, II, and III. 7044


Research Article

ACS Catalysis

Figure 9. Selected representative structures of the active site in the initial hydrolytic reaction.

Figure 10. Evolution of selected interatomic distances along the hydrolysis reaction.

Figure 11. (A) The predicted relative free-energy profiles along the hydrolysis reaction by QM(B3LYP)/MM MD simulations. (B) The distance change of the binuclear zinc center along the hydrolysis reaction.

Znα···Oμ, Znβ···Oμ, and P···Oμ are 2.00, 1.96, and 4.03 Å, respectively. The hydrolytic reaction proceeds to the first transition state (TS1) with a free barrier of 9.8 kcal/mol, in which the P···Oμ distance is remarkably shortened from ∼4.03 to 2.16 Å, while the P−F bond length is less changed (see Figure 10). In addition, the Znβ···Oμ bond length increases to 2.24 Å, and the OL···Znβ distance and the bond angle of P···OL···Znβ decrease to 2.05 Å and 123°, respectively, compared to corresponding distances of 1.96 and 2.90 Å and the angle of

153° in the reactant conformation, as shown in Figures 9, 10 and Table S1. Once the reaction passes through TS1, the intermediate (Int) is formed with an energy release of 2.2 kcal/mol, in which the Znβ···μ−OH bond is almost broken with a large distance of 2.73 Å, and the OL···Znβ bond (1.95 Å) is formed completely. We note that the Znα···Znβ and P···F distances increase to 4.0 and 1.75 Å at this stage, leading to a metastable precursor to the dissociation of F−, as shown in Figures 9−11. The reaction 7045


Research Article

ACS Catalysis

Figure 12. Representative structures and conformational changes of the active domain during the F− release process.

phosphate obtained by Kim et al.5 Presumably, the dissociation and release of the degraded phosphate from the active site is probably difficult. 3.4. Release of Fluorion. Our QM/MM MD simulations reveal that the number of water molecules around the fluorion increases gradually with the dissociation of F− from the active site (see Figure S2), which leads to a first solvation shell of six water molecules for the fluorion, as shown in Figure 12. Here the P···F distance was defined as the reaction coordinate (RC3) and used in computation of the free-energy profile for the F− release. As Figure 12 shows, the fluorion is totally located in the bulk water as RC3 reaches to 9.2 Å, and the F− release is a barrier-free process with an energy release of 6.6 kcal/mol (see Figure 13), which may facilitate the development and sustainability of the hydrolytic reaction. 3.5. Release of the Degraded Product. The predicted relative free-energy profiles for the hydrolytic reaction steps and the fluorion release suggest that this enzymatic detoxification should have high efficiency. However, the enzymatic activity against the degradation of sarin is much lower than that in hydrolysis of paraoxon by PTE.29,25 We note that with the fluorion release, the degraded product, a phosphoester, is bridged to both zinc sites in a bidentate coordination mode, in which Znα keeps the pentacoordinate structure, while Znβ binds two water molecules and becomes hexa-coordinate. At the product-bound state, dissociation and release of the degraded product are likely difficult, even though the leaving group is exposed to the solvent. Considering the stable bidentate bound state of the degraded product and a complex local environment, we investigated different possibilities for its dissociation and release by using QM/MM MD simulations with the umbrella sampling, a stateof-the-art approach to simulating enzymes, in combination with

Figure 13. Relative free-energy profiles for the fluorion release.

proceeds to TS2 with a free-energy barrier of 12.3 kcal/mol relative to the initial reactant, in which the P···F distance is 2.06 Å and the Asp301···H distance is continuously shortened to 1.39 Å. As the hydrolysis proceeds further, the F− anion will leave away from the organophosphate substrate, coupled with the proton transfer from the μ−OH to the residue Asp301. The freeenergy span for the two-step hydrolytic chemical process is 12.3 kcal/mol. As Figure 11A shows, the process is remarkably favorable, both dynamically and thermodynamically, and furthermore the solvation of F− in bulk water may facilitate its release from the active domain. At the product state, the degraded product symmetrically bridges two divalent zinc ions through Oμ···Znα (2.00 Å) and OL···Znβ (1.95 Å) bonding interactions (see Table S1 and Figure 9), as observed in the Xray crystal structure of complex of PTE with the diethyl 7046


Research Article

ACS Catalysis

Figure 14. Possible mechanisms for release of the degraded product considered in four different models A−D, in which the residue Asp301 maintains the protonated state at this stage.

Figure 16. Predicted relative free-energy profiles for dissociation of the degraded product in Model D by QM(B3LYP)/MM MD simulations. Figure 15. Reaction coordination (RC7) for the product dissociation in Model D.

Figures S6, S10, and S12 in the Supporting Information, and thus, they are impossibly involved in the enzymatic detoxification of sarin. Alternatively, Model D provides a practicable channel to release of the final degraded product, in which the proton transfer from the water molecule to the binuclear zincbound degraded product (recombination), the bridging coordination of the nascent OH− (stabilization), and the dissociation of the neutral product from the active site

MM MD simulations. Figure 14 depicts four plausible models used in the exploration of the degraded product release. Our QM/MM calculations and MD simulations reveal that release of the degraded product in Models A, B, and C experiences substantial high free-energy barriers, as shown in 7047


Research Article

ACS Catalysis

Figure 17. Representative structures and conformational changes of the active domain in PTE during the dissociation and release of the degraded product in Model D.

degraded product and surrounding water molecules. The product release is approximately divided into three stages, as shown in Figures 17 and S14, and the diameter of the entrance channel reaches its maximum value of about 20.5 Å in Stage II, showing that the substrate is just passing through the channel. As the degraded product leaves away from the active site, the delivery channel restores its normal state in Stage III. We note that the multiple chemical steps are involved in the low-energy dissociation pathway of the binuclear Zn2+-bound degraded product, and they dominate the detoxification efficiency of PTE toward sarin, while the hydrolysis of P−F bond is relatively facile. Previous studies suggest that the enzymatic kinetic parameters for the hydrolysis of substituted esters depend on the pKa values of the leaving groups.60,61 For the substrates with a leaving group of pKa > 7, the cleavage of phosphate P−O bond is rate limiting. On the contrary, for those pKa < 7, the product release is likely dominant for the enzymatic hydrolysis.38 Here the fluorion leaving group has the pKa value

(dissociation) are involved (see Figure 14). In order to describe the multistep dissociation process, the reaction coordinate (RC7), defined by the combination of several selected distances (see Figure 15), was used in QM/MM MD simulations for the potential of mean force. As Figure 16 shows, the recombination process, including the proton transfer, the conversion from the bidentate to monodentate coordination, and the formation of the bridginghydroxide group, experiences a free-energy barrier of 21.0 kcal/ mol. The subsequent ligand substitution of one water molecule for the phosphoester product has a free-energy barrier of 14.8 kcal/mol, and this ligand exchange follows a SN2 mechanism, as shown in Figures 17 and S13. The overall dissociation process is endothermic. We note that the solvation of phosphoester in bulk water can bring about a stabilization energy of about 25.3 kcal/ mol with release of the degraded product, which may drive the product release from the active site. As Figure 17 shows, there are relatively strong hydrogen bonding interactions between the 7048


Research Article

ACS Catalysis of 3.2, and the cleavage of P−F bond is indeed not the ratedetermining step, which lends support to this dependence of the pKa-value on the enzymatic hydrolysis.

4. CONCLUSIONS Extensive QM(DFT)/MM MD and MM MD simulations have been used to explore the whole journey of the enzymatic detoxification for the G-type nerve agent sarin by PTE, including the substrate delivery to the active site, the catalytic reaction, and the release of products. Here both divalent zinc ions in the active site are bridged by the carboxylate group (Lys169) and the nucleophile hydroxide ion, and the coordination of two water molecules to Znα and Znβ, respectively, resulting in the sixcoordinated metal centers in the ready state of PTE. RAMD-MD simulations reveal that the channel Pa, along the leaving pocket, is basically responsible for the substrate delivery to the active site with an energy release of 16.8 kcal/mol. The enzymatic P−F cleavage of the substrate sarin follows a two-step mechanism, in which the nucleophilic attack of the bridging hydroxide leads to the P−Oμ formation with the free-energy barrier of 9.8 kcal/mol, and followed by the P−F cleavage and the proton transfer from the μ−OH to the residue Asp301, the degraded product is formed with the free-energy span of 12.3 kcal/mol. After the F− release, the degraded product is tightly bound to the binuclear zinc center in the bidentate coordination, and its dissociation is predicted to be rate-determining for the enzymatic efficiency. The multiple chemical steps are involved in dissociation of the degraded product, and the predicted free-energy barriers for the low-energy pathway are 21.0 kcal/mol for the recombination step and 18.3 kcal/mol for dissociation of the neutral product. The solvation of products in the bulk solvent is generally exothermic remarkably, which may drive the release of products. The present work provides a comprehensive understanding of the enzymatic detoxification of sarin by PTE, which is important for further experimental studies and the enzyme engineering of degrading toxic organophosphorus compounds.

■

■

coordinates defined in Models C and D (Figure S11); The predicted free-energy profiles for dissociation and release of the degraded product in Models C and D (Figure S12); Evolution of selected interatomic distances along dissociation and release of the degraded product in Model D (Figure S13); Diameter fluctuation of the transportation channel characterized by the distance between Leu271 and Phe132 along three stages for release of the degraded product. (Figure S14) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.C). *E-mail: [email protected] (J.Z). ORCID

Zexing Cao: 0000-0003-0803-7732 Author Contributions §

F.F. and Y.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (21873078, 21673185, and 21373164).

■

REFERENCES

(1) Benning, M. M.; Hong, S. B.; Raushel, F. M.; Holden, H. M. The Binding of Substrate Analogs to Phosphotriesterase. J. Biol. Chem. 2000, 275, 30556−30560. (2) Zhan, D.; Zhou, Z.; Guan, S.; Han, W. The Effect of Conformational Variability of Phosphotriesterase upon N-Acyl-LHomoserine Lactone and Paraoxon Binding: Insights from Molecular Dynamics Studies. Molecules 2013, 18, 15501−15518. (3) Bigley, A. N.; Mabanglo, M. F.; Harvey, S. P.; Raushel, F. M. Variants of Phosphotriesterase for the Enhanced Detoxification of the Chemical Warfare Agent VR. Biochemistry 2015, 54, 5502−5512. (4) Tsai, P. C.; Fan, Y.; Kim, J.; Yang, L.; Almo, S. C.; Gao, Y. Q.; Raushel, F. M. Structural Determinants for the Stereoselective Hydrolysis of Chiral Substrates by Phosphotriesterase. Biochemistry 2010, 49, 7988−7997. (5) Kim, J.; Tsai, P. C.; Chen, S. L.; Himo, F.; Almo, S. C.; Raushel, F. M. Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphodiesterase. Biochemistry 2008, 47, 9497−9504. (6) Goldsmith, M.; Eckstein, S.; Ashani, Y.; Greisen, P.; Leader, H.; Sussman, J. L.; Aggarwal, N.; Ovchinnikov, S.; Tawfik, D. S.; Baker, D.; Thiermann, H.; Worek, F. Catalytic Efficiencies of Directly Evolved Phosphotriesterase Variants with Structurally Different Organophosphorus Compounds in Vitro. Arch. Toxicol. 2016, 90, 2711−2724. (7) Wolfenden, R. Degrees of Difficulty of Water-Consuming Reactions in the Absence of Enzymes. Chem. Rev. 2006, 106, 3379− 3396. (8) Petrović, D.; Szeler, K.; Kamerlin, S. C. L. Challenges and Advances in the Computational Modeling of Biological Phosphate Hydrolysis. Chem. Commun. 2018, 54, 3077−3089. (9) Castro, A. A. De; Assis, L. C.; Silva, D. R.; Corrêa, S.; Assis, T. M.; Gajo, G. C.; Soares, F. V.; Ramalho, T. C. Computational Enzymology for Degradation of Chemical Warfare Agents: Promising Technologies for Remediation Processes. AIMS Microbiol. 2017, 3, 108−135. (10) Manco, G.; Porzio, E.; Suzumoto, Y. Enzymatic Detoxification: A Sustainable Means of Degrading Toxic Organophosphate Pesticides and Chemical Warfare Nerve Agents. J. Chem. Technol. Biotechnol. 2018, 93, 2064−2082. (11) Yang, Y. C. Chemical Detoxification of Nerve Agent VX. Acc. Chem. Res. 1999, 32, 109−115.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.9b01877. Evolution of the zinc-water distance during the 200 ns MD simulation (Figure S1); The statistic number of water molecules around the F−ion during its release from the active site (Figure S2); Representative structures and conformational changes of the active domain during the product release in Model A (Figure S3); Evolution of ZnαO2 and Znα-water distances during the product release in Model A (Figure S4); Free-energy profiles and the statistic number of water molecules around Znα and Znβ during dissociation and release of the degraded product in Model A (Figure S5); The defined reaction coordinates for the “hydroxylation”, “exchange” and “dissociation” steps in Model B (Figure S6); The predicted relative energy profiles along six reaction coordinates for the “exchange” step in Model B (Figure S7); Representative structure of the active domain during the product release process in Model B (Figure S8); Representative structures of the active domain during the product release process in Model B (Figure S9); The predicted relative energy profiles for the “hydroxylation”, “exchange”, and “dissociation” steps in Model B (Figure S10); The reaction 7049


Research Article

ACS Catalysis

Detoxifying Enzymes Using Computationally Focused Libraries. ACS Chem. Biol. 2013, 8, 2394−2403. (32) Bigley, A. N.; Desormeaux, E.; Xiang, D. F.; Bae, S. Y.; Harvey, S. P.; Raushel, F. M. Overcoming the Challenges of Enzyme Evolution to Adapt Phosphotriesterase for V-Agent Decontamination. Biochemistry 2019, 58, 2039−2053. (33) Johansson, K. E.; Lindorff-Larsen, K. Structural Heterogeneity and Dynamics in Protein Evolution and Design. Curr. Opin. Struct. Biol. 2018, 48, 157−163. (34) Bigley, A. N.; Xiang, D. F.; Ren, Z.; Xue, H.; Hull, K. G.; Romo, D.; Raushel, F. M. Chemical Mechanism of the Phosphotriesterase from Sphingobium Sp. Strain TCM1, an Enzyme Capable of Hydrolyzing Organophosphate Flame Retardants. J. Am. Chem. Soc. 2016, 138, 2921−2924. (35) Benning, M. M.; Kuo, J. M.; Raushel, F. M.; Holden, H. M. Three-Dimensional Structure of Phosphotriesterase: An Enzyme Capable of Detoxifying Organophosphate Nerve Agents. Biochemistry 1994, 33, 15001−15007. (36) de Castro, A. A.; Caetano, M. S.; Silva, T. C.; Mancini, D. T.; Rocha, E. P.; da Cunha, E. F. F.; Ramalho, T. C. Molecular Docking, Metal Substitution and Hydrolysis Reaction of Chiral Substrates of Phosphotriesterase. Comb. Chem. High Throughput Screen. 2016, 19, 334−344. (37) Vernekar, A. A.; Das, T.; Mugesh, G. Vacancy-Engineered Nanoceria: Enzyme Mimetic Hotspots for the Degradation of Nerve Agents. Angew. Chem., Int. Ed. 2016, 55, 1412−1416. (38) Wong, K.; Gao, J. The Reaction Mechanism of Paraoxon Hydrolysis by Phosphotriesterase from Combined QM/MM Simulations The Reaction Mechanism of Paraoxon Hydrolysis by Phosphotriesterase From. Biochemistry 2007, 46, 13352−13369. (39) Ghanem, E.; Raushel, F. M. Detoxification of Organophosphate Nerve Agents by Bacterial Phosphotriesterase. Toxicol. Appl. Pharmacol. 2005, 207, 459−470. (40) Zhao, Y.; Cao, Z. X. Global Simulations of Enzymatic Catalysis. Acta Phys. - Chim. Sin. 2017, 33, 691−708. (41) Vanhooke, J. L.; Benning, M. M.; Raushel, F. M.; Holden, H. M. Three-Dimensional Structure of the Zinc-Containing Phosphotriesterase with the Bound Substrate Analog Diethyl 4-Methylbenzylphosphonate. Biochemistry 1996, 35, 6020−6025. (42) Rostkowski, M.; Olsson, M. H.; Søndergaard, C. R.; Jensen, J. H. Graphical Analysis of pH-Dependent Properties of Proteins Predicted Using PROPKA. BMC Struct. Biol. 2011, 11, 6−12. (43) Case, D. A.; Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A. Development and Testing of a General Amber Force Field. J. Comput. Chem. 2004, 25, 1157−1174. (44) Gao, Y.; Zhang, C.; Wang, X.; Zhu, T. A Test of AMBER Force Fields in Predicting the Secondary Structure of A-Helical and B-Hairpin Peptides. Chem. Phys. Lett. 2017, 679, 112−118. (45) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A WellBehaved Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges: The RESP Model. J. Phys. Chem. 1993, 97, 10269−10280. (46) Wang, J.; Cieplak, P.; Kollman, P. How Well Does a Restrained Electrostatic Potential(RESP) Model Perform in Calculating Conformational Energies of Organic and Biological Molecules? J. Comput. Chem. 2000, 21, 1049−1074. (47) Frisch, M. J.; W. Trucks, G.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;

(12) Benschop, H. P.; De Jong, L. P. A. Nerve Agent Stereoisomers: Analysis, Isolation, and Toxicology. Acc. Chem. Res. 1988, 21, 368−374. (13) Dawson, R. M. Review of Oximes Available for Treatment of Nerve Agent Poisoning. J. Appl. Toxicol. 1994, 14, 317−331. (14) Chambers, J. E.; Oppenheimer, S. F. Organophosphates, Serine Esterase Inhibition, and Modeling of Organophosphate Toxicity. Toxicol. Sci. 2004, 77, 185−187. (15) Goldsmith, M.; Aggarwal, N.; Ashani, Y.; Jubran, H.; Greisen, P.; Ovchinnikov, S.; Leader, H.; Baker, D.; Sussman, J. L.; Goldenzweig, A.; Fleishman, S. J.; Tawfik, D. S. Overcoming an Optimization Plateau in the Directed Evolution of Highly Efficient Nerve Agent Bioscavengers. Protein Eng. Des. Sel. 2017, 30, 333−345. (16) Millard, C. B.; Kryger, G.; Ordentlich, A.; Greenblatt, H. M.; Harel, M.; Raves, M. L.; Segall, Y.; Barak, D.; Shafferman, A.; Silman, I.; Sussman, J. L. Crystal Structures of Aged Phosphonylated Acetylcholinesterase: Nerve Agent Reaction Products at the Atomic Level. Biochemistry 1999, 38, 7032−7039. (17) Quinn, D. M. Acetylcholinesterase: Enzyme Structure, Reaction Dynamics, and Virtual Transition States. Chem. Rev. 1987, 87, 955− 979. (18) Raushel, F. M. Bacterial Detoxification of Organophosphate Nerve Agents. Curr. Opin. Microbiol. 2002, 5, 288−295. (19) Lundebye, A. K.; Curtis, T. M.; Braven, J.; Depledge, M. H. Effects of the Organophosphorous Pesticide, Dimethoate, on Cardiac and Acetylcholinesterase (AChE) Activity in the Shore Crab Carcinus Maenas. Aquat. Toxicol. 1997, 40, 23−36. (20) Stone, R. How to Defeat a Nerve Agent. Science 2018, 359, 23− 23. (21) Ojha, A.; Gupta, Y. K. Evaluation of Genotoxic Potential of Commonly Used Organophosphate Pesticides in Peripheral Blood Lymphocytes of Rats. Hum. Exp. Toxicol. 2015, 34, 390−400. (22) Yu, C. J.; Du, J. C.; Chiou, H. C.; Chung, M. Y.; Yang, W.; Chen, Y. S.; Fuh, M. R.; Chien, L. C.; Hwang, B.; Chen, M. L. Increased Risk of Attention-Deficit/Hyperactivity Disorder Associated with Exposure to Organophosphate Pesticide in Taiwanese Children. Andrology 2016, 4, 695−705. (23) Zhang, P.; Liu, E. J.; Tsao, C.; Kasten, S. A.; Boeri, M. V.; Dao, T. L.; DeBus, S. J.; Cadieux, C. L.; Baker, C. A.; Otto, T. C.; Cerasoli, D. M.; Chen, Y. T.; Jain, P.; Sun, F.; Li, W.; Hung, H.; Yuan, Z.; Ma, J.; Bigley, A. N.; Raushel, F. M.; Jiang, S. Nanoscavenger Provides LongTerm Prophylactic Protection against Nerve Agents in Rodents. Sci. Transl. Med. 2019, 11, eaau7091. (24) Bigley, A. N.; Raushel, F. M. The Evolution of Phosphotriesterase for Decontamination and Detoxification of Organophosphorus Chemical Warfare Agents. Chem.-Biol. Interact. 2019, 308, 80−88. (25) Tsai, P. C.; Bigley, A.; Li, Y.; Ghanem, E.; Cadieux, C. L.; Kasten, S. A.; Reeves, T. E.; Cerasoli, D. M.; Raushel, F. M. Stereoselective Hydrolysis of Organophosphate Nerve Agents by the Bacterial Phosphotriesterase. Biochemistry 2010, 49, 7978−7987. (26) Zhang, X.; Wu, R.; Song, L.; Lin, Y.; Lin, M.; Cao, Z.; Wu, W.; Mo, Y. Molecular Dynamics Simulations of the Detoxification of Paraoxon Catalyzed by Phosphotriesterase. J. Comput. Chem. 2009, 30, 2388−2401. (27) Omburo, G. A.; Kuo, J. M.; Mullins, L. S.; Raushel, F. M. Characterization of the Zinc Binding Site of Bacterial Phosphotriesterase. J. Biol. Chem. 1992, 267, 13278−13283. (28) Caldwell, S. R.; Newcomb, J. R.; Schlecht, K. A.; Raushel, F. M. Limits of Diffusion in the Hydrolysis of Substrates by the Phosphotriesterase from Pseudomonas Diminuta. Biochemistry 1991, 30, 7438−7444. (29) Tsai, P. C.; Fox, N.; Bigley, A. N.; Harvey, S. P.; Barondeau, D. P.; Raushel, F. M. Enzymes for the Homeland Defense: Optimizing Phosphotriesterase for the Hydrolysis of Organophosphate Nerve Agents. Biochemistry 2012, 51, 6463−6475. (30) Bigley, A. N.; Mabanglo, M. F.; Harvey, S. P.; Raushel, F. M. Variants of Phosphotriesterase for the Enhanced Detoxification of the Chemical Warfare Agent VR. Biochemistry 2015, 54, 5502−5512. (31) Cherny, I.; Greisen, P.; Ashani, Y.; Khare, S. D.; Oberdorfer, G.; Leader, H.; Baker, D.; Tawfik, D. S. Engineering V-Type Nerve Agents 7050


Research Article

ACS Catalysis Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.1.; Gaussian, Inc.: Wallingford, CT, 2009. (48) Sun, Y.; Kollman, P. A. Hydrophobic Solvation of Methane and Nonbond Parameters of the TIP3P Water Model. J. Comput. Chem. 1995, 16, 1164−1169. (49) Simmerling, C.; Cheatham, T. E.; Wang, B.; Case, D. A.; Merz, K. M.; Darden, T.; Gohlke, H.; Onufriev, A.; Woods, R. J.; Luo, R. The Amber Biomolecular Simulation Programs. J. Comput. Chem. 2005, 26, 1668−1688. (50) Kollman, P. A.; Miyamoto, S. SETTLE: An Analytica; Version of the SHAKE and RATTLE Algorithm for Rigid Water Models. J. Comput. Chem. 1992, 13, 952−962. (51) Kumar, S.; Rosenberg, J. M.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A. Mu1ti Dimensional Free-Energy Calculations Using the Weighted Histogram Analysis Method. J. Comput. Chem. 1995, 16, 1339−1350. (52) Roux, B. The Calculation of the Potential of Mean Force Using Computer Simulations. Comput. Phys. Commun. 1995, 91, 275−282. (53) Zhang, Y.; Liu, H.; Yang, W. Free Energy Calculation on Enzyme Reactions with an Efficient Iterative Procedure to Determine Minimum Energy Paths on a Combined Ab Initio QM/MM Potential Energy Surface. J. Chem. Phys. 2000, 112, 3483−3492. (54) Neese, F. Software Update: The ORCA Program System, Version 4.0. WIREs Comput. Mol. Sci. 2018, 8, e1327. (55) Kalé, L. V.; Bhandarkar, M.; Brunner, R.; Krawetz, N.; Phillips, J.; Shinozaki, A. NAMD: A Case Study in Multilingual Parallel Programming. Lect. Notes Comput. Sci. 1998, 1366, 367−381. (56) Yu, L. J.; Golden, E.; Chen, N.; Zhao, Y.; Vrielink, A.; Karton, A. Computational Insights for the Hydride Transfer and Distinctive Roles of Key Residues in Cholesterol Oxidase. Sci. Rep. 2017, 7, 1−13. (57) Fan, F.; Chen, N.; Wang, Y.; Wu, R.; Cao, Z. QM/MM and MM MD Simulations on the Pyrimidine-Specific Nucleoside Hydrolase: A Comprehensive Understanding of Enzymatic Hydrolysis of Uridine. J. Phys. Chem. B 2018, 122, 1121−1131. (58) Zhao, Y.; She, N.; Ma, Y.; Wang, C.; Cao, Z. A Description of Enzymatic Catalysis in N-Acetylhexosamine 1-Kinase: Concerted Mechanism of Two-Magnesium-Ion-Assisted GlcNAc Phosphorylation, Flexibility Behavior of Lid Motif upon Substrate Recognition, and Water-Assisted GlcNAc-1-P Release. ACS Catal. 2018, 8, 4143−4159. (59) Zhao, Y.; Chen, N.; Wang, C.; Cao, Z. A Comprehensive Understanding of Enzymatic Catalysis by Hydroxynitrile Lyases with S Stereoselectivity from the α/β-Hydrolase Superfamily: Revised Role of the Active-Site Lysine and Kinetic Behavior of Substrate Delivery and Sequential Product Release. ACS Catal. 2016, 6, 2145−2157. (60) Aubert, S. D.; Li, Y.; Raushel, F. M. Mechanism for the Hydrolysis of Organophosphates by the Bacterial Phosphotriesterase. Biochemistry 2004, 43, 5707−5715. (61) Cleland, W. W.; Hengge, A. C. Enzymatic Mechanisms of Phosphate and Sulfate Transfer. Chem. Rev. 2006, 106, 3252−3278.

7051


A Comprehensive Understanding of Enzymatic Degradation of the G

Recommend Documents