Insights into the Stereospecificity in Papain-Mediated

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Articles Cite This: ACS Chem. Biol. 2019, 14, 1280−1292

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Insights into the Stereospecificity in Papain-Mediated Chemoenzymatic Polymerization from Quantum Mechanics/ Molecular Mechanics Simulations Joan Gimenez-Dejoz, Kousuke Tsuchiya, and Keiji Numata* Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Downloaded via 95.85.68.113 on August 29, 2019 at 00:32:58 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Chemoenzymatic peptide synthesis is an efficient and clean method to generate polypeptides for new applications in the fields of biomedical and functional materials. However, this enzyme-mediated synthesis is dependent on the reaction rate of the protease biocatalyst, which is essentially determined by the natural substrate specificity of the enzyme. Papain, one of the most studied cysteine proteases, is extensively used for the chemoenzymatic synthesis of new polypeptides. Similar to most proteases, papain displays high stereospecificity toward L-amino acids, with limited reactivity for the Dstereoisomer counterparts. However, the incorporation of D-amino acids into peptides is a promising approach to increase their biostability by conferring intrinsic resistance to proteolysis. Herein, we determined the stereospecific-limiting step of the papainmediated polymerization reaction with the chiral substrates L/D-alanine ethyl ester (Ala-OEt). Afterward, we used Quantum Mechanics/Molecular Mechanics (QM/MM) simulations to study the catalytic mechanism at atomic level of detail and investigate the origin of its stereospecificity. The experimental and computational results show that papain is able to attack both L- and D-stereoisomers of Ala-OEt, forming an enzyme−substrate intermediate, and that the two reactions display a similar activation barrier. Moreover, we found that the reduced catalytic activity of papain in the polymerization of D-amino acids arises from the aminolysis step of the reaction, in which L-Ala-OEt displays a significantly lower free-energy barrier (12 kcal/mol) than D-Ala-OEt (30 kcal/mol). Further simulations suggest that the main factor affecting the polymerization of D-amino acids is the configuration of the D-acyl-intermediate enzyme, and in particular the orientation of its methyl group, which hinders the nucleophilic attack by other monomers and thus the formation of polypeptides.

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INTRODUCTION

Chemoenzymatic peptide synthesis has emerged as an effective alternative for the production of peptides to chemical and bacterial syntheses.9,10 The biochemical reactions involved in chemoenzymatic synthesis require protease enzymes as a biocatalyst. The main advantages of this technique are its high yield, atom-economy, regio- and stereoselectivity, lack of requirement for protection of the amino acid side chains, and mild reaction conditions.11,12 In contrast, the most widely used method in chemical synthesis of peptides, solid-phase peptide synthesis (SPPS), requires multiple protection and depro-

There is an increasing interest in the use of polypeptides because of their diverse physical properties and biological functions. Their versatile applications include pharmaceuticals,1 therapeutic biomedical applications, such as drugdelivery systems,2,3 and nutrition as food-derived bioactive compounds.4 Another relevant area attracting interest is the use of engineered polypeptides as green, biobased innovative alternatives to petroleum-derived materials. Polypeptides are the building blocks of protein materials, such as spider silk, which exhibit high mechanical proprieties thanks to their secondary structure and hierarchical arrangement, which are lacking in the majority of synthetic polymer materials.5−8 © 2019 American Chemical Society

Received: April 3, 2019 Accepted: May 7, 2019 Published: May 7, 2019 1280

DOI: 10.1021/acschembio.9b00259 ACS Chem. Biol. 2019, 14, 1280−1292

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ACS Chemical Biology

Figure 1. Reaction scheme of the chemoenzymatic peptide synthesis mediated by papain. In the first step, acylation, formation of the acylintermediate enzyme occurs after release of a leaving molecule from the substrate. In the acyl-intermediate stage, both aminolysis and hydrolysis reactions may happen, depending on nucleophiles present in the reaction.

tection steps of the lateral chains as well as toxic chemicals.13,12 Another extensively used chemical method, polymerization of α-amino acid-N-carboxyanhydrides (NCA), can achieve synthesis of high-molecular-weight polypeptides, but the technique limitations, the high reactivity of NCA prone to side reactions, establish that only homopolymers, random copolypers, or graft copolymers without sequence specificity can be synthesized.14,15 Although it is well-known that proteases catalyze the cleavage of the peptide bonds, chemoenzymatic peptide synthesis takes advantage that, under specific conditions, proteases can catalyze the formation of peptide bonds.16−18 This ability reflects the fact that enzymes do not alter the thermodynamic equilibrium of the reactions; therefore, they can catalyze both hydrolysis and aminolysis reversibly. Under physiological conditions, this equilibrium is strongly turned to the direction of hydrolysis.19,20 Therefore, in order to use proteases, the reaction equilibrium must be adjusted to promote the formation of the amide bond via aminolysis. This can be accomplished by two different methods, namely, thermodynamic controlled synthesis (TCS) or kinetically controlled synthesis (KCS).17,21 In KCS, which is the most widely used technique, a serine or a cysteine protease performs a nucleophilic attack at the carbonyl carbon atom of the acyl donor substrate, which has to be activated in ester, amide, or nitrile form (e.g., alanine ethyl ester (Ala-OEt)). This chemical reaction produces the enzyme−substrate complex, in which the substrate is covalently bonded with the protease, resulting in an acylenzyme intermediate after the release of a leaving molecule, such as an ethanol molecule from Ala-OEt. At this point, the acyl-intermediate enzyme is deacylated by the attack of a nucleophile present in the medium. If this nucleophile is the amino group of an amino acid monomer, bond formation (aminolysis) will occur, resulting in the fusion of the monomers into a polypeptide. However, if the attacking nucleophile is a water molecule, hydrolysis takes place instead (Figure 1).13,17 Thus, in KCS, a competition between aminolysis and hydrolysis reactions takes place in the deacylation step.17 This competition implies that a high concentration of alternative nucleophiles to water must be present in the reaction mixture for aminolysis to occur. When the concentration of the alternative nucleophile decreases during peptide polymerization, the rate of the hydrolysis reaction overcomes that of aminolysis, resulting in the undesired hydrolysis of the synthesized polypeptide. The success of the aminolysis step thus depends on fundamental reaction parameters, such as the concentrations and ratio of enzyme and substrate,22,23 pH,9,24 temperature,13,25 and substrate specificity of the enzyme.17,25 Therefore, the reaction

rate of the polymerization reaction is basically determined by the specificity of the enzyme for the acyl donor.13 Papain (EC 3.4.22.2), a cysteine protease, has been extensively used in chemoenzymatic peptide synthesis.9,11,26−28 It presents a catalytic dyad formed by the residues Cys25 and His159.29,30 The imidazole group of His159 polarizes the thiol group of the Cys25, deprotonating it and forming an ion-pair dyad,31,32 while the near Asn175 residue stabilizes His159 but is not essential for the catalysis.33 Papain presents a broad substrate specificity and is able to polymerize L-amino acids into polypeptides, produce telechelic peptides34 as well as recognize and polymerize some unnatural amino acids (e.g., incorporating nylon units with different amino acid monomers).35 However, despite its broad specificity, papain cannot efficiently hydrolyze or catalyze aminolysis using D-amino acids as substrates. Previous studies by Schechter et al. found that papain exhibited reduced catalytic efficiency in the hydrolysis of a polyalanine peptide containing one D-alanine unit.36 Beer et al., using mimetic substrates as carboxybenzyl protected amino acids incorporating a guanidinophenyl ester group (OGp),26,27 showed that papain could polymerize unnatural amino acids, such Z-D-Ala-OGp, with an attacking L-Phe-NH2 nucleophile to form a dipeptide, but the formation of higherorder polymers was precluded. However, when the monomer was changed to D-Phe-NH2, the reaction shifted to hydrolysis. A recent study showed that 2-aminoisobutyric acid (Aib), an unnatural amino acid similar to alanine, was not polymerized by papain, either in its monomeric (Aib-OEt) or dipeptide (Aib-Ala-OEt) ethyl ester forms. The papain-catalyzed polymerization proceeded only when the Aib unit was surrounded by L-alanines (AlaAibAla-OEt), showing again the preferential substrate specificity of the enzyme for the Lamino acids.28 Another striking example of the enantioselectivity of papain is its ability to efficiently polymerize L-Ala-OEt with different ester groups, but no polymerization is observed when D-Ala-OEt (or D-alanine with different ester groups) is used as substrate.9,23 However, D-amino acid containing peptides represent attractive building blocks for the design of peptide-based functional biomaterials and therapeutic agents, because the D-amino acids can stabilize or α-helix structures or promote novel secondary structures. In addition, their recognition by proteases is more difficult, protecting the biomaterial against proteolytic degradation by endogenous enzymes, which increases its biostability and pharmacological availability.37−42 Several computational studies have investigated the hydrolysis reaction of peptide bonds by cysteine proteases, including papain.43−49 However, to the best of our knowledge, no previous study has explored the formation of peptide bonds 1281


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or D-Ala-OEt for the polymerization assays was washed three times with Milli-Q water and lyophilized. The lyophilized samples were diluted in DMSO-d6/TFA-d (5:1) for 1H NMR analysis. Nuclear Magnetic Resonance and Optical Rotatory Dispersion Measurements. 1H NMR spectra were recorded on a Varian NMR system 500 (500 MHz) spectrometer (Varian Medical Systems, Palo Alto, CA) at 25 °C controlled with the VnmrJ software. Sixty-four scans were taken during each NMR experiment. Data were processed and analyzed by ACD/NMR Processor (academic Edition) software (v.12.01, Advanced Chemistry Development, Inc.). Optical rotatory dispersion (ORD) measurements of the L- and D-Ala-OEt samples (10 mg mL−1) were recorded in 1,1,1,2,2,2-hexafluoroisopropanol/trifluoroacetic acid (TFA) (4;1, v/v) at 20 °C, using a Jasco J-820 spectropolarimeter at wavelengths from 300 to 700 nm. Model Preparation and Classical Molecular Dynamics Simulations. The initial structure of papain used in the simulations was obtained from the reported X-ray structure of the enzyme (PDB ID: 1PPN, resolution: 1.60 Å).53 This structure consists of one chain with 212 amino acids, together with a bound atom ligand and a methanol molecule that were removed from the structure. The protonation states of amino acid residues were set according to previous studies: in particular, the catalytic His159 residue was protonated while Cys25 was deprotonated, forming a zwitterionic ionpair dyad in the model of the acylation reaction.32,45,49,54 For the aminolysis reaction, the His159 residue was kept in neutral εprotonated state, and the acyl-intermediate was constructed according to the procedure described in the Supporting Information (Acylintermediate models construction). The constructed papain models were treated using AMBER ff14SB force field,55 while the L- and D-Ala-OEt substrates were described using the GAFF force field,56 and the overall system was solvated with TIP3P water molecules.57 A time step of 2 fs was used, along with the SHAKE algorithm,58 while the particle-mesh Ewald (PME)59 method was used for the calculation of long-range interactions. The systems were equilibrated by a series of gradual-step simulations, in which distance restraints were set between the substrate molecules and protein to maintain important contact interactions. Concretely, in the acylation reaction, contacts between the oxygen atom of the carbonyl of the Ala-OEt ligands and the oxyanion hole, formed by the backbone NH of Cys25 and lateral chain of Gln19, were maintained because they are thought to play an important role in the catalytic activity of papain30 and other cysteine proteases.54 The simulations were carried out for 10 ns, after which the system was considered to be equilibrated according to the root-mean-square deviation (RMSD) of the enzyme heavy atoms (Figure S1). Relaxed structures selected from the last part of the MD trajectories were used as starting structures for hybrid QM/MM calculations described in the following sections. Analysis of trajectories was done using CPPTRAJ60 and VMD software.61 Further details of the classical MD simulations are described in the section Computational details of Supporting Information. QM/MM Molecular Dynamics Simulations. Hybrid QM/MM MD calculations at high level of theory were performed using the DFT B3LYP/6-31G* basis set62 for the QM atoms. The QM calculations were carried out using the external Gaussian0963 program, together with the AMBER/Gaussian interface.64 In this scheme, the dynamics of the atoms treated with MM were described with the AMBER ff14SB force field. The integration time step for the QM calculations was set to 0.2 fs. The explicit link atom approach implemented in sander, the MD engine of the AMBER code, was used to separate the QM and the MM regions when their boundary crossed covalent bonds.65 The electrostatic interactions between MM and QM regions were truncated at a cutoff of 8 Å.64 All results reported on this work are from QM/MM at a high level of theory. Adaptively Biased Molecular Dynamics. The FEL of the acylation and aminolysis reactions were determined using ABMD.50,51 This enhanced sampling technique, based on the metadynamics approach,52,66 introduces a time-evolving biasing potential in the simulation, enabling the exploration of energy surfaces of selected collective variables (CVs). The CV must be able to discriminate

via aminolysis. In addition, no study has focused on understanding the stereospecificity that papain, as the majority of proteases, displays toward L- and D-amino acids. The limited understanding of the molecular basis of the aminolysis reaction and its stereospecificity currently hinders the development of enzyme-mediated chemistry and biotechnologies. Therefore, in this study, we tried to understand how the chemoenzymatic polymerization is accomplished via papain-catalyzed aminolysis at atomic level of detail. Here, we performed papain-mediated chemoenzymatic peptide synthesis reactions using L- and D-Ala-OEt stereoisomers as substrates, to determine the enzyme stereospecificity. 1H NMR analysis of the reactions was used to reveal the rate-limiting step of D-Ala-OEt in the chemoenzymatic polymerization reaction with papain. To get further insight into the reaction mechanism, we used quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations to investigate, at atomistic level of detail, the papain-catalyzed acylation and aminolysis reactions of L- and D-Ala-OEt, and determine their free-energy landscapes (FELs). In particular, we employed the adaptively biased molecular dynamics (ABMD) method,50,51 based on metadynamics,52 to enhance the sampling efficiency of the simulations, together with the first-principle density functional theory (DFT) framework. The experimental and computational results reveal that papain can recognize both L- and DAla-OEt stereoisomers in the acylation step, forming an acylintermediate enzyme with a similar energy barrier, and demonstrate that the different reactivity of the enzyme toward the Ala-OEt stereoisomers arises from the aminolysis step of the polymerization reaction. This information would open the way to the rational design of more efficient protease enzymes to be used as biocatalyst, with broadened specificity for the efficient polymerization of unnatural and D-amino acids to generate new peptides with enhanced biostability.

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EXPERIMENTAL SECTION

Materials. L-Ala-OEt, D-Ala-OEt, and L-alanine tert-butyl ester (LAla-OtBu) hydrochlorides were purchased from Sigma-Aldrich. Deuterium oxide was purchased from Cambridge Isotope Laboratories, Inc. Papain (EC No. 3.4.22.2), derived from the unripe fruit of the papaya tree (Carica papaya), was purchased from Wako Pure Chemical Industries. Standard chemicals were purchased from Wako Chemical Co. Papain activity was approximately 0.086 ± 0.005 U mg−1, where one unit of activity is defined as the amount of enzyme required to hydrolyze and release 1 μmol of substrate, fluorescein isothiocyanate (FITC), from FITC-labeled casein, per min at 25 °C pH 7.5 (protease fluorescent detection kit, Sigma-Aldrich). Chemoenzymatic Polymerization Assays. The polymerization reactions with L-Ala-OEt, D-Ala-OEt, or L-Ala-OtBu were based on the reaction conditions of previous studies.23 Briefly, the reactions were conducted in an EYELA Chemistation with stirring at 800 rpm at 40 °C in 25 mL glass reaction tubes with a final volume of 5 mL in 1 M phosphate buffer pH 8.0, containing 1 U ml−1 of papain. For chemoenzymatic assays with different feed ratio, a final concentration of 1 M substrate was used, but varying the ratio of L- and D-Ala-OEt. A concentration of 0.6 M for D-Ala-OEt and L-Ala-OtBu was used for the polymerization reactions analyzed by 1H NMR. The reaction was carried out for 3 h for D-Ala-OEt and L-Ala-OtBu 1H NMR assays and 2 h for L- and D-Ala-OEt polymerization assays. Control reactions were performed without adding enzyme to the assay. After the reaction times listed above, the reactions mixtures were centrifuged at 10 000g for 10 min to remove precipitated papain or products. The supernatant of the reactions with D-Ala-OEt or L-Ala-OtBu for NMR assays was mixed with D2O at a final concentration of ∼10 mg mL−1 in NMR glass tubes (5 mm). The precipitate of the reactions with L1282


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distance of C1Acyl‑intermediateN1Ala‑OEt (b). CV2 was defined as a LCOD between N1Ala‑OEtH1Ala‑OEt (c) minus the H1Ala‑OEt HδHis159 distance (d) (Figure 2b). The first (CV1) describes the attack of the nucleophile to the acyl-intermediate C1 atom and the breaking of Cys25-Sγ and C1 atom bond. The second (CV2) describe the proton transfer from the amino group of the nucleophile to His159.

between the different states of the system (reactants, products, intermediates, etc.), describe all the key events, and drive the reaction.66 The flooding time scale of the bias deposition was set at 30 fs (150 MD steps), and the resolution was set to 1 kcal/mol. First crossing criterion was used to determine the simulation end, as it is recommended for chemical reactions.67 QM/MM ABMD Simulations of the Acylation Reaction. To check the feasibility of the CVs, FELs were first explored using a semiempirical QM level of theory (details in Supporting Information, Semiempirical QM/MM ABMD simulations and Figure S2). Then, we performed QM/MM ABMD simulations at higher level of theory (DFT B3LYP/6-31G* basis set). A total of 47 and 53 (including 4 H link atoms) atoms were treated quantum mechanically for L/D-AlaOEt and L-Ala-OtBu, respectively. Two CVs (CV1 and CV2) were used to describe the acylation reaction. Both CVs are described as a linear combination of distances (LCOD) corresponding to bond breaking minus bond forming in the reaction. CV1 was defined as the LCOD between the C1Ala‑OEtO1Ala‑OEt distance (a) minus the SγCys25C1Ala‑OEt distance (b). Whereas CV2 was the LCOD of NδHis159HδHis159 distance (c) minus HδHis159O1Ala‑OEt distance (d) (Figure 2a). Maximum distances at 3.60 Å between the reactant or product, Cys25 and His159 were used to limit the FEL space of the chemical event and thus reduce the computational effort.

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RESULTS AND DISCUSSION Experimental Evaluation of D-Ala-OEt Reactivity with Papain. A mixture of D- and L-Ala-OEt at different feed ratios was reacted with papain to evaluate its stereospecific reactivity. Polymerization of the monomers only occurred in the reactions that presented a feed ratio equal or with higher proportion of L-Ala-OEt (Table S1 and Figure S3). On the other hand, the reaction yield decreased with increasing D-AlaOEt fraction, and no polymerization was observed when the feed ratio of D-Ala-OEt monomer was higher than L-Ala-OEt. The polypeptides obtained from reactions with a mixed feed of monomers were analyzed by ORD to evaluate their chirality and estimate if they were made pure or mixed L- and Dmonomers. They showed a decrease in their specific rotation [α]d compared with the polymers produced by the reaction with pure L-Ala-OEt. This suggest that, in the polymerization reactions with a low feed ratio of D-Ala-OEt, some Dmonomers could be polymerized and incorporated into the polypeptide (Table S1). As expected,23 these results confirmed that papain is not able to polymerize pure D-Ala-OEt, but suggest that some D-amino acids can be polymerized by papain when L-Ala-OEt coexists in the reaction. It is widely accepted that the reaction mechanism of proteases involves two steps: formation of the acylintermediate and either the hydrolysis with water molecules or aminolysis with substrates. To determine which step is ratelimiting in the polymerization of D-Ala-OEt, we used 1H NMR to characterize the reaction product of papain with pure D-AlaOEt. If papain cannot recognize D-Ala-OEt as a substrate, the 1 H NMR spectra will show only the peaks corresponding to the monomer present in the solution. On the other hand, if papain reacts with D-Ala-OEt and form and acyl-intermediate, in the reaction solution spectra we should find the peaks corresponding to hydrolyzed D-Ala-OEt (namely, D-alanine) and ethanol. Indeed, the results show the presence of D-alanine and ethanol in the NMR spectra of the reaction solution, with

Figure 2. Representation of the atoms treated at the B3LYP/6-31G* QM level of theory for acylation (a) and aminolysis (b) reactions. Link atoms between the QM and MM regions are displayed in curly lines. Blue arrows mark the CV1 a and b distances, while black arrows show the CV2 c and d distances. QM/MM ABMD Simulations of the Aminolysis Reaction. The aminolysis was explored through the same procedure described for the acylation reaction in the previous section, except for the number of treated QM atoms and CVs. In the aminolysis reaction, a total of 58 atoms (including 4 H link atoms) were treated QM. The following CVs were used to model the reaction. CV1 was taken as the LCOD between the distance of SγCys25C1Acyl‑intermediate (a) minus the

Figure 3. 1H NMR spectra of the reaction products of D-Ala-OEt using papain as biocatalyst (a) and of the control reaction carried out without enzyme (b). 1283


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Figure 4. Free energy landscape of the papain-catalyzed acylation reaction of L-Ala-OEt (a) and D-Ala-OEt (b), calculated using QM/MM simulations at the B3LYP/6-31G* level of theory. Time evolution of the CV distances along the reaction for L- (c) and D-Ala-OEt (d). R, reactants; TS, transition state; AI, acyl-intermediate. Isolines are drawn at 2 kcal/mol intervals.

both stereoisomers, the CVs were able to reproduce the acylation reaction with a single concerted mechanism, driving the system from the enzyme/substrate reactants to the acylintermediate enzyme, and producing an ethanol molecule. The acylation reaction was then modeled using QM/MM MD simulations at a higher level of QM theory (B3LYP/6-31G*). Starting structures for QM/MM simulations with L- and D-AlaOEt were selected from the final (equilibrated) classical MD trajectories after careful examination (Figure S6). The set of CVs illustrated in Figure 2a was used to drive the reactants (R, consisting of the papain enzyme and substrate in reactive conformation) to form the acyl-intermediate enzyme (AI), releasing an ethanol molecule. As the experiments showed, papain is able to form an acylintermediate enzyme with both L- and D-Ala-OEt. To obtain more details of the papain-mediated acylation and quantify possible differences between the stereoisomers, we modeled the reactions with both the stereoisomers by QM/MM and determined their free-energy barriers. The FEL of the acylation reaction performed with L- and D-Ala-OEt as substrates is presented in Figures 4a,b and S7. The reactants (R) and the acyl-intermediate (AI) correspond to the two minima located on the lower-left and upper-right corners of the FEL, respectively. No energy minimum separating the reactants and acyl-intermediate is found in the FELs, indicating that both substrates undergo concerted reactions, with only two energy minima separated by one transition state (TS). An activation barrier of 23 kcal/mol to reach the TS is found for the reaction of L-Ala-OEt (Figure 4a). The process starts

an estimated conversion of D-Ala-OEt hydrolysis of 48.3%, given that the integration of the NMR peak areas was 48.3% (Figure 3a, peak H over A). However, only the peaks corresponding to D-Ala-OEt were observed in the negative control reaction carried out without the enzyme (Figure 3b). The presence of ethanol and free D-alanine in the first solution provides evidence of the hydrolysis of D-Ala-OEt performed by the enzyme. Therefore, papain recognizes both alanine ethyl ester stereoisomers in the acylation step, forming an acylintermediate enzyme. This means that the different reactivity of the enzyme toward both stereoisomers thus arises from the second step of the reaction (i.e., aminolysis). To confirm this point, we performed the same reaction using a similar molecule, L-Ala-OtBu, which also cannot be polymerized by papain.22 The 1H NMR spectrum of the reaction product of LAla-OtBu with papain does not display significant differences compared with that of the control reaction without enzyme (Figure S4). Only some minor peaks, which might suggest a trace presence of the hydrolyzed product, are visible in the spectrum. Hence, L-Ala-OtBu is not recognized by papain and remains unreactive, unable to form the acyl-enzyme intermediate, and therefore is neither hydrolyzed or polymerized. This different behavior shows that although D-AlaOEt can enter the acylation reaction and form an acylintermediate with papain, the subsequent aminolysis cannot proceed. QM/MM Simulation of the Acylation Reaction. The FELs obtained from the semiempirical QM simulations to assess the suitability of the CVs are shown in Figure S5. For 1284


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Figure 5. Representative snapshots of the mechanism of the acylation reaction of L-Ala-OEt (a−d) and D-Ala-OEt (e−h) extracted from the lowest free-energy pathway calculated with the QM/MM ABMD simulations. Papain is represented as a gray cartoon, while atoms QM-treated are illustrated using ball-and-stick representations. Carbon atoms for L- and D-Ala-OEt are displayed in green and blue color, respectively.

Ala-Ala-Ala-Gly-Ala-OCH3).69 Moreover, the computed FEL is in accordance with other studies46,49 that predicted that the acylation reaction follows a concerted mechanism, in which the transfer of the proton does not precede the attack and covalent bond formation between the Cys25-Sγ atom and the substrate. However, at variance with a previous report,49 in our case no stable intermediate is found in the acylation reaction using a similar DFT setup. A different mechanism, as the authors of the previous study reported, could be explained due to the different chemical structure of the substrates used, enzyme environment, computational methods, or different levels of theory.49,70 The experimentally obtained free-energy barriers of the hydrolysis of p-nitrophenyl esters and p-anisidides by papain (pH 6.0 and 35 °C), in which the acylation step was determined to be the rate-limiting step, were previously estimated to be 16.9 and 17.9 kcal/mol, respectively, using transition state theory.71 However, it should be considered that the reaction rates of cysteine proteases are dependent on substrates and reaction conditions, such temperature.71 The above QM/MM simulations of the acylation reaction were started from structures in which the oxygen atom of the carbonyl group of the substrates faced the oxyanion hole of the catalytic site of papain. It has been suggested that the oxyanion hole, formed by the backbone NH group of the Cys25 residue and the side chain of Gln19, stabilizes the TS and reduces the activation energy of the reaction. In addition, early site directed mutagenesis studies with papain showed that the mutation of Gln19 to alanine or serine affects the kcat value of the reaction, suggesting the existence of hydrogen bonds between Gln19 and the substrate in the transition state.30,72 Notably, these contacts were observed in the present simulations with both stereoisomers, which occur when the oxygen atom of the carbonyl group of the Ala-OEt substrate is stabilized by Hbond interactions with both the Gln19 and the NH group of the main chain of Cys25 (Figure S9). To understand the effect of oxyanion hole on the energy barrier of the reaction, we modeled the acylation reaction of

with the distance between the Cys25-Sγ atom and the C1 atom of the substrate being reduced. Concurrently, the H atom of the protonated His159 moves closer to the O1 of the ester group of the substrate (Figures 4c and 5b). This step is followed by C1−O1 bond breaking in ester group (CV1 a) and the proton transfer from His159 (CV2 c) (Figure 5c) via a downward pathway of 27 kcal/mol and the formation of the acyl-intermediate enzyme, in which a stable covalent bond is formed between the Cys25-Sγ atom and the C1 atom of the substrate carbonyl group. Correspondingly, the proton of the His159 is transferred to the O1 of the ethyl ester group, forming an ethanol molecule (Figure 5d). Hence, even if the Cys25-Sγ attack and bond formation with the substrate slightly precedes the proton transfer from His159 (Figures 4c and 5a− d), they are practically synchronous. Similarly, the FEL of the reaction of D-Ala-OEt substrate (Figure 4b) displays a TS 19 kcal/mol above local minimum of the reactants. Thus, the 4 kcal/mol difference with respect to LAla-OEt shows that the substrate stereospecificity has little effect on the enzyme activity in the acylation reaction. This difference may be attributed to the interaction between the lateral chain of D-Ala-OEt and Gly23 residue, while the L-AlaOEt lateral chain is close but not in contact with the main chain of His159. D-Ala-OEt undergoes the same synchronous process described above for L-Ala-OEt, in which the Cys25-Sγ attack occurs slightly earlier than the proton transfer from the His159 residue to the O1 atom to form the ethanol molecule (Figures 4d, 5e−h, and S8). Even if the predicted activation energy for L-Ala-OEt is slightly overestimated, the activation energies obtained for the acylation reactions are in agreement with previous studies by Wei et al. (19.8 kcal/mol with papain and N-acetyl-Phe-Gly 4nitroanilide as substrate),46 Elsässer et al. (19.3 kcal/mol with human legumain and cystatin shortened pentamer),68 Ma et al. (19.8 kcal/mol in human cathepsin K with an Ace-Leu-ArgPhe-Nme substrate),54 and Arafet et al. (24.8 kcal/mol in cruzain protein from Trypanosoma cruzi and the peptide Ac1285


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Figure 6. FEL obtained from the QM/MM simulations at DFT level of theory (a,d), time evolution of CVs along the reaction (b,e), and enlarged view of CV evolution for 1−2 ps before product formation (c,f), for the aminolysis reaction with L-Ala-OEt (top) and D-Ala-OEt (bottom). AI, acyl-intermediate; I, intermediate; TS, transition state. Isolines are drawn at 2 kcal/mol intervals. Green shadowed area corresponds to the intermediate.

single TS and two energy minima corresponding to the reactants and the acyl-intermediate (Figure S14). The analysis of the trajectory reveals that the bulkier tert-butyl group of the substrate, compared with ethyl ester, precludes the correct configuration of L-Ala-OtBu into the active site due to steric hindrance on close adjacent residues, especially Gln19 and Trp177, that prevents the substrate from reaching a favorable position near the catalytic Cys25. In particular, one methyl group of the tert-butyl ester group is sandwiched between and sterically interferes with the Gln19 and Trp177 residues. This forces Gln19 to move very close to the main backbone chain of the enzyme, disrupting the contacts of the substrate with the oxyanion hole and laterally displaces Trp177 to the other side of the active site cleft of papain. The different positioning of the Ala-OEt and Ala-OtBu substrates is reflected in the distance between the Gln19 oxygen atom and the Trp177 NH group during the simulations (Figure S15). In the acylation reaction with L-Ala-OEt, the average distance until the formation of the acyl-intermediate is 2.7 Å, whereas separation increases to 4.9 Å in the reaction with L-Ala-OtBu. As indicated above, in the reaction performed with L-Ala-OtBu, no trace of the hydrolyzed substrate or tert-butanol could be detected by 1 H NMR (Figure S4), indicating that papain is not reactive with L-Ala-OtBu. This unreactive behavior of this substrate can be explained by the high-energy barrier calculated by QM/MM simulations of the acylation for L-Ala-OtBu monomer as well as the mechanistic insights at atomic detail obtained. Thus, this finding highlights the advantages of the combination of experimental and QM/MM approaches to obtain the accurate acylation reaction model. QM/MM Simulation of Aminolysis Reaction. Similar to acylation, aminolysis reaction was initially modeled at a lower level of QM theory. The corresponding models and calculated FELs are shown in Figures S2b and S16. The FELs of the aminolysis reactions of L- and D-Ala-OEt substrates, obtained

the D-Ala-OEt substrate starting from a configuration in which the oxygen atom of its carbonyl group was not located in the oxyanion hole. In this simulation, the initial distances between the oxygen atom of the carbonyl group of D-Ala-OEt and the NH groups of Gln19 and Cys25 were 4.7 and 4.8 Å, respectively (Figure S10). As a result, the free-energy barrier for the acylation reaction increased considerably, reaching 35 kcal/mol (Figure S11). This is 14 kcal/mol higher compared with the energy barrier of the D-Ala-OEt reaction carried out with the oxygen atom initially located near the oxyanion hole (Figure 4b). In addition, the reaction only took place when the oxygen atom of the carbonyl group closely approached the oxyanion hole, forming H-bonds with its atoms (Figure S12). The interaction between the Cys25 NH group and the oxygen atom of the carbonyl group of the acyl-intermediate, which are found at a mean distance of ∼2.3 Å for all simulations after the acyl-intermediate formation, seems to be particularly important for the stabilization of the intermediate (Figures S9b and S12b). The 1H NMR analysis spectra of the reaction of L-Ala-OtBu with papain discussed in the section Experimental Evaluation of d-Ala-OEt Reactivity with Papain suggested that papain does not polymerize or hydrolyze L-Ala-OtBu, indicating that the enzyme cannot form the acyl-intermediate with this monomer (Figure S4). Therefore, we also performed QM/ MM simulations of the acylation reaction with L-Ala-OtBu (Figure S13), to confirm that the activation barrier for this reaction is higher than that for the reactions of L- and D-AlaOEt. Accordingly, the FEL obtained for the acylation of L-AlaOtBu reveals a free-energy barrier of 30 kcal/mol for the formation of the acyl-intermediate (Figure S13); as expected, this value is 7 and 11 kcal/mol higher compared to the barriers obtained for L- and D-Ala-OEt, respectively. The acylation proceeds in a similar way to that observed for the Ala-OEt monomers, that is, through a concerted reaction involving a 1286


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Figure 7. Representative snapshots of the mechanism of the aminolysis reaction of L-Ala-OEt (a−d) and D-Ala-OEt (e−h) extracted from the lowest free-energy pathway calculated with the QM/MM ABMD simulations. Papain is represented as a gray cartoon, while atoms QM-treated are illustrated using ball-and-stick representations. Carbon atoms for L- and D-Ala-OEt are displayed in green and blue color, respectively.

intermediate slightly precedes the proton transfer of the amino group of the substrate to His159. In contrast with the concerted mechanism predicted by the L-Ala-OEt reaction mentioned above, the reaction with D-AlaOEt follows a concerted mechanism; moreover, no intermediate can be observed in the FEL calculated for the D-AlaOEt reaction (Figure 6d). The reaction requires the H1 of the amino group of the D-Ala-OEt to be located at a short distance (∼1.6 Å) from the Nδ atom of the His159 (CV2 d in Figure 6e,f). Afterward, the N1 atom of the amino group of the nucleophile attacks the C1 atom of the acyl-intermediate, reducing their distance to ∼1.6 Å (Figure 6f). The distance between Cys25-Sγ and C1 of acyl-intermediate (CV1 a) starts increasing at this stage and continuously increases until the TS is formed. In the reaction with L-Ala-OEt, this distance increases to ∼2.1 Å, forming the shortly lived intermediate, and after that, it continues to increase forming the product. In the case of D-Ala-OEt, the torsion angle of the complex formed by the amino group of the attacking D-Ala-OEt nucleophile and the C1 atom of the D-acyl-intermediate becomes strained (Figure 7f); the close proximity between the methyl groups of the acyl-intermediate and the D-Ala-OEt substrate produces a steric hindrance that must be overcome for the attack to occur. A straining on the angle geometry the amino group of the attacking D-Ala-OEt nucleophile and the C1 atom of the Dacyl-intermediate enables the N1 atom of the substrate to attack the C1 atom, which is concealed by the methyl group of the acyl-intermediate. Hence, an increase in the free energy for the reaction is observed. Finally, immediately after the reaction, the methyl groups of the newly formed D-Ala-AlaOEt rapidly separate, acquiring a more relaxed configuration without steric hindrance (Figure 7h). Some key interactions are also different in the reactions of the two stereoisomers. For example, the oxygen atom of the acyl-intermediate in the reaction with D-Ala-OEt overlaps with

from the QM/MM ABMD simulations at a high level of theory are shown in panel a and d, respectively, of Figure 6 (Figures S17 and S18). In contrast with the acylation reaction, the shape of the FEL of the aminolysis reaction with L-Ala-OEt (Figure 6a) indicates a stepwise mechanism, with a small local minimum (corresponding to an intermediate species: I) located between reactants (AI; acyl-intermediate enzyme and L-Ala-OEt acting as nucleophile) and products (P; L-Ala-AlaOEt and regenerated enzyme) (Figure 6a). This intermediate shows an ΔG basin of ∼1 kcal/mol and therefore is highly unstable. The computed free-energy barrier for the reaction was 12 kcal/mol. Importantly, this energy is markedly lower (16 kcal/mol) than that obtained for the D-Ala-OEt stereoisomer (28 kcal/mol, Figure 6d). Furthermore, the FEL profile of the reaction of D-Ala-OEt denotes a concerted mechanism, without the intermediate state observed for the aminolysis of LAla-OEt, even though this intermediate is unstable due to its small activation energy. As discussed above, the aminolysis reaction of L-Ala-OEt, follows a stepwise mechanism with a short-lived intermediate. First, the amino group of L-Ala-OEt moves closer to the C1 atom of the acyl intermediate (with CV1 b decreasing to ∼2.6 Å). Concurrently, the H1 atom of L-Ala-OEt moves closer to the Nδ of His159 (CV2 c), establishing and maintaining a close contact with it (∼1.9 Å). When the distance between the C1 atom of the acyl-intermediate and the N1 atom of the substrate (CV1 b) becomes shorter than 2.0 Å, the distance between the Cys25-Sγ and C1 atom of the acyl-intermediate (CV1 a) increases from ∼1.8 to 2.1 Å, leading to the small intermediate seen in the FEL (Figures 6a and 7b,c). Then, the distance between C1 and N1 (CV1 b) atoms continues to decrease, and the H1 proton of the amino group is rapidly transferred to His159 (Figure 7c), yielding an L-Ala-Ala-OEt molecule and the regenerated enzyme (Figure 7d). Thus, the attack of the N1 atom of the nucleophile to the C1 atom of the acyl1287


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Figure 8. Computed free-energy landscape of the aminolysis reaction with papain (a,b), and time evolution of distances corresponding to the selected CVs (c,d) in the L-acyl-intermediate + D-Ala-OEt (LD) (a,c) and D-acyl-intermediate + L-ala-OEt (DL) (b,d) reactions. AI; acylintermediate. I, intermediate; TS, transition state; P, products. Isolines are drawn at 2 kcal/mol intervals. The green shadowed area corresponds to the intermediate.

of D-Ala-OEt near the Nδ of His159, and the attack of the N1 amino group to the acyl-intermediate C1 atom. In contrast, in the aminolysis reaction with L-Ala-OEt, the methyl groups of the nucleophile point in the opposite direction and do no sterically interfere with the acyl-intermediate, since its closest atom to the L-Ala-OEt amino group is a hydrogen (Figure 7c). The comparison of the energy barriers obtained for the acylation and aminolysis reactions suggests that the ratelimiting step for L-Ala-OEt is the acylation stage, which has a 11 kcal/mol higher activation barrier than the aminolysis (Table S2). In contrast, when D-Ala-OEt is used as the substrate, the limiting step that precludes the reaction to occur is the aminolysis, in agreement with the experimental results (Figure 3), with an energy barrier of 28 kcal/mol (9 kcal/mol higher than for acylation) (Table S2). Interestingly, the activation energy for the acylation reaction with L-Ala-OtBu, which is not able to react with papain (Figure S4) and therefore does not form an acyl-intermediate enzyme, has a similar activation energy of 30 kcal/mol (Figure S14, Table S2). Recently, in a computational study, Elsässer et al. reported energetics of the transpeptidation reaction in the human legumain enzyme.68 The reaction was found to proceed via a concerted mechanism, with a transition state energy of 23.3 kcal/mol. In the same study, the authors also reported a freeenergy barrier to hydrolysis of 7.1 kcal/mol and noted that hydrolysis is preferred when a water molecule is present in the reaction site. Other studies have predicted free-energy barriers

the Cys25 residue of the papain, due to the configuration of the attacking nucleophile. This is seen in the average distance between the oxygen atom of the acyl-intermediate and the NH of the Cys25 in the oxyanion hole, that is, shorter in the D-AlaOEt (∼2.2 Å) than L-Ala-OEt (∼2.5 Å) reaction (Figure S19), although during the intermediate (L-Ala-OEt) and TS (D-AlaOEt), the distance is ∼2.1 Å in both cases. Further differences are observed in the distance between the Gln19 residue and the oxygen of the carbonyl group of the acyl-intermediate. This distance is longer (∼2.4 Å) and exhibits wider fluctuations in the aminolysis reaction of D-Ala-OEt than L-Ala-OEt (∼1.9 Å) (Figure S19b). A similar trend is observed for the distance between the oxygen atom of the carbonyl group of the substrate and the H-bond formed with the nearby Trp177 residue, which helps to position and stabilize L-Ala-OEt in the active-site (Figure S20). For the D-Ala-OEt substrate to attack the acyl-intermediate, the ethyl ester group of the nucleophile molecule needs to be oriented toward and above the Gln19. Consequently, the loop preceding the α-helix where Cys25 is located moves and slightly changes its position. Consequently, the position of the Gly23 residue in the loop is affected, and no H-bond is formed between the oxygen of the main chain of Gly23 and the amino group of the acyl-intermediate. Instead, the amino group of the acyl intermediate is located nearby the H of the Gly23, resulting in a weaker interaction and thus a higher activation energy (Figure S21). The geometrical features discussed above hinder the correct position of the H1 atom of the amino group 1288


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ACS Chemical Biology of the acyl-enzyme hydrolysis in papain of 10.7 kcal/mol46 and 10−14 kcal/mol.49 These energies are very similar to those calculated for aminolysis with L-Ala-OEt (12 kcal/mol) in the present study (Table S2). This suggests that a competition between the aminolysis and hydrolysis reactions occur, as shown by the present experiments. In contrast, the reaction with D-Ala-OEt presents a much higher energetic barrier (28 kcal/mol) than the hydrolysis of the acyl-intermediate. Consequently, the D-Ala-OEt substrate cannot efficiently compete with the water molecules. Because of its more energetically favorable pathway and the presumably higher abundance of water molecules, hydrolysis predominates over the aminolysis reaction with the D-Ala-OEt substrate. Reactions with Different Stereoisomers for Substrate and Acyl-Intermediate. To further understand how the geometries of the attacking nucleophile and acyl-intermediate affect the aminolysis reaction based on the experimental polymerization results with different feed ratios of L- and D-AlaOEt monomers (Table S1), we performed two additional QM/MM ABMD simulations, in with we varied the substrate stereoisomer acting as nucleophile in the aminolysis step. In the first simulation, we modeled the reaction (labeled LD) of the L-acyl-intermediate, produced from the acylation reaction with L-Ala-OEt, with D-Ala-OEt as nucleophile (Figure S22a); in the second simulation (labeled LD), the D-acyl-intermediate, generated from the acylation reaction with D-Ala-OEt, was reacted with an L-Ala-OEt attacking molecule (Figure S22b). Therefore, with this notation, the simulations of the aminolysis reaction discussed above can be labeled LL for the reaction of the L-acyl-intermediate with L-Ala-OEt as nucleophile, and DD for the reaction between the D-acyl-intermediate and D-AlaOEt as attacking nucleophile (Table S3). The LD aminolysis reaction shows a similar reaction mechanism to the LL one, with a short-lived intermediate formed by the attack of the nucleophile along with the increased length and small stabilization of the bond between the Cys25-Sγ and the C1 atom of the acyl-intermediate, before the proton transfer (Figure 8a and Figures S23−25). The freeenergy barrier is calculated to be 15 kcal/mol, which is 3 kcal/ mol higher than that of the LL aminolysis and 13 kcal/mol lower than that computed for the DD reaction (Table S3). In contrast, the aminolysis reaction with the opposite conformation (DL) displays a free-energy barrier of 22 kcal/mol and, as previously observed for the DD aminolysis, no intermediate state is observed in the FEL (Figures 6d and 8b). The computed energy barrier for the DL reaction is thus 10 kcal/mol higher than that LL aminolysis but still 6 kcal/mol lower compared with the aminolysis involving D-Ala-OEt alone (DD). The above activation free energies indicate that the geometry of the acyl-intermediate is the main factor affecting the outcome of the aminolysis reaction. Changing the nucleophile (from L- to D-Ala-OEt) while keeping the acylintermediate in the L-configuration results in an increase of 3 kcal/molof the kinetic barrier. Therefore, the stereochemical configuration of the Ala-OEt nucleophile accounts for only a small portion of the energy barrier. On the other hand, changing the stereochemical configuration of the acylintermediate from L to D, increases the activation barrier by at least 10 kcal/mol (compare the DL to LL values in Table S3). In this regard, only the reactions with an L-acyl-intermediate enzyme presented a small intermediate state in the reaction (Figures 6a and 8a), confirming that the strained configuration

of the D-acyl-intermediate observed in the reactions is less stable and that the reaction rapidly proceeds to products. This is due to the lower steric hindrance of the attacking nucleophile, in a similar way as for the LL aminolysis, since its methyl group is oriented in a way that only makes steric contact with a hydrogen atom of the acyl-intermediate (rather than with a methyl group, as in the case with D-acylintermediate) (Figure S25e-g). Thus, the methyl group of the D-acyl-intermediate hinders the attack by both L- and DAla-OEt nucleophiles, preventing the favorable positioning and attack of the C1 atom of the carbonyl group by the amino group of the nucleophile. The ORD experimental results suggested that the polymerization reactions using a combination of L- and D-Ala-OEt with papain occur and that some polymerized peptides incorporate a D-Ala-OEt monomer (Table S1). The simulation results indicate that the aminolysis with D-Ala-OEt nucleophile take place, even though it presents higher energetic barriers than LAla-OEt. These newly polymerized peptides incorporating Dalanine, in the next polymerization cycle, produce D-acylenzyme-intermediate that hinders the attack of the next monomer, thereby increasing the energy barrier for the aminolysis (Table S3). According to the experimental result (Figure 3) and the calculated energy barriers, D-acylintermediate can react only with L-Ala-OEt monomer, even though with higher energy barrier compared with an LL reaction. The chemoenzymatic polymerization reactions with low D-monomer feed ratio can proceed and produce peptides with a small content of D-Ala-OEt, although their yield is much lower in comparison with the LL reactions. This could be because the D-acyl-intermediate is more easily hydrolyzed than L-acyl-intermediate because of the lower energy barrier for hydrolysis (10−14 kcal/mol) compared with the DD and DL reactions (28 and 22 kcal/mol respectively). Furthermore, the incorporation of the next L-Ala-OEt monomer to D-acylintermediate is more energetically expensive (22 kcal/mol) than the aminolysis with a L-acyl-intermediate (12 kcal/mol) (Table S3). Two consecutive D-Ala-OEt monomers cannot be polymerized, according to the calculated energy barriers.

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CONCLUSIONS To clarify the molecular recognition of protease in chemoenzymatic polymerization of amino acids, we have investigated the papain-mediated acyl-intermediate formation, hydrolysis, and aminolysis using L- and D-Ala-OEt. According to the present experiments, the different stereospecificity of papain originates from the aminolysis reaction. Then, we employed enhanced-sampling QM/MM simulations at the DFT B3LYP/ 6-31G* level of theory to gain insight in the mechanism. The simulations show that the acylation step proceeds through a concerted mechanism for both isomers, whose reactions display similar activation energies. In the case of aminolysis reaction with L-Ala-OEt, the free-energy barriers of the aminolysis and hydrolysis reactions are comparable (12 kcal/ mol), which makes the L-Ala-OEt nucleophile capable of competing with water in the deacylation of the acylintermediate. On the other hand, the aminolysis with D-AlaOEt has to overcome the steric hindrance produced by the methyl group of the acyl-intermediate on the attacking D-AlaOEt, which increases the activation barrier of the reaction by 16 kcal/mol compared with L-Ala-OEt. Additional simulations carried out with different combinations of the two stereoisomers as acyl-intermediates and nucleophiles reveal that the 1289



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orientation of the methyl group of the D-acyl-intermediate is the main cause of its different reactivity, with free-energy barriers for aminolysis consistently higher than those of the Lacyl-intermediates. The residues involved in stabilizing the acyl-intermediate and the nucleophile in the aminolysis, and therefore reducing the activation barrier, seem to play a similar paper with both stereoisomers. The only exception is Gly23, which displays more stable contacts with the L-Ala-OEt stereoisomer. Overall, the results of the QM/MM simulations are consistent with the present experiments and provide the detailed atomic-scale mechanism in the polymerization reaction. Therefore, on the basis of our findings, one can conceive a rational modification of proteases focused on changes that favor alternative configurations for the D-acylintermediate, in order to reduce the energy barrier required for the aminolysis. These can favor the design of protease enzymes with broadened stereospecificity that are able to easily polymerize D- or unnatural amino acids. Increasing the range and availability of biocatalysts to incorporate D-amino acids is an essential step to fully develop enzymatic-mediated chemistry, especially to create more biostable peptides as therapeutic drugs and to explore new biobased materials with structural/mechanical features over L-amino acid-based ones.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.9b00259.

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Computational details (System Preparation and Classical Molecular Dynamics Simulations, Acyl-intermediate models construction, and Semiempirical QM/MM ABMD simulations); Table S1−S3 and Figures S1− S25 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Keiji Numata: 0000-0003-2199-7420 Author Contributions

J.G.-D. and K.N. conceived and designed the research. J.G.-D. performed the experiments and simulations and analyzed the data. K.T. also performed experiments related to aminolysis. J.G.-D. wrote a draft, and K.T. and K.N. edited the manuscript. All authors reviewed the manuscript. Funding

This work was supported by Technology Agency Exploratory Research for Advanced Technology (JST-ERATO; Grant No. JPMJER1602). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge S. Nakamura, K. Ogata, and Y. Sakamoto for their help in setting the QM/MM simulations. We acknowledge RIKEN ACCC for providing access to Hokusai/GreatWave and BigWaterfall supercomputing resources. 1290


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