Deacylation Mechanism and Kinetics of AcylâEnzyme Complex of

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Deacylation Mechanism and Kinetics of Acyl–Enzyme Complex of Class–C #-Lactamase and Cephalothin Ravi Tripathi, and Nisanth N. Nair J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b11623 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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The Journal of Physical Chemistry

Deacylation Mechanism and Kinetics of Acyl–Enzyme Complex of Class–C β -Lactamase and Cephalothin Ravi Tripathi and Nisanth N. Nair∗ Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, India E-mail: [email protected]

KEYWORDS: Class-C β-Lactamase, Antibiotic Resistance, Deacylation, Reaction Mechanism, QM/MM, Metadynamics

∗

To whom correspondence should be addressed

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ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Understanding the molecular details of antibiotic resistance by the bacterial enzymes β–lactamases is vital for the development of novel antibiotics and inhibitors. In this spirit, the detailed mechanism of deacylation of the acyl–enzyme complex formed by cephalothin and class–C β–lactamase is investigated here using hybrid quantum– mechanical/molecular–mechanical molecular dynamics methods. Roles of various active site residues and substrate in the deacylation reaction are elucidated. We identify the base that activates the hydrolyzing water molecule and the residue that protonates the catalytic serine (Ser64 ). Conformational changes in the active sites and proton transfers that aid to efficiently carry out the deacylation reaction are presented. We have also characterized the oxyanion–holes and other H–bonding interactions that stabilize the reaction intermediates. Together with the kinetic and mechanistic details of the acylation reaction, we analyze the complete mechanism and the overall kinetics of the drug hydrolysis. Finally, the apparent rate–determining step in the drug hydrolysis is scrutinized.

Introduction Bacterial infections are becoming increasingly untreatable as a result of growing antibiotic resistance in bacteria posing a serious threat to the public health. 1–11 One of the most common resistance mechanisms adopted by bacteria is the deactivation of β–lactam antibiotics by its reaction with bacterial enzymes called β–lactamases. β-lactamases catalyze irreversible hydrolytic opening of the β-lactam ring of the drug molecules. 12–14 Relentlessly growing antibiotic resistance raises the fear of going back to the pre-antibiotic era when most of the available antibiotics would become ineffective for the treatment of infectious diseases. 6 Among the four classes of β-lactamases, 15 our focus of this study is the Class–C βlactamases (CBL). These enzymes are mainly found in gram negative bacteria 16,17 and gained attention due to their ability to hydrolyze the third–generation cephalosporins and carbapen2


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R

OH2

N O H

R

R

OH2

B

O

O

HN

N

B

O

O

O

Ser64

Ser64

HB

TI ES−EI

ES

H

H

EI

R

O

O

Ser64

R O HN

HO OH

HN B

O

OH HB

Ser64

EP

Ser64

TI EI−EP

Figure 1: General mechanism of acylation and deacylation (in dotted box) steps of a β-lactam antibiotic with CBL. Here B indicates a basic residue, which could be either an active site residue or a part of the drug molecule itself. ems. 18,19 CBL catalyze the drug hydrolysis in two steps 20–28 (see also Figure 1 ): (a) acylation process, which involves β–lactam ring–opening and formation of a covalent complex with the enzyme; (b) deacylation reaction, composed of hydrolysis of the drug–enzyme covalent bond, thus reforming the enzyme active site and releasing the hydrolyzed drug. Understanding the molecular level details of both these processes is of paramount significance in designing novel antibiotics and inhibitors. 28 The active site residues of CBL that are believed to play crucial roles during the catalysis are Ser64 , Lys67 , Tyr150 , Asn152 , Ala220 and Lys315 29–34 (See FigSI 1) Among these, the precise roles of Lys67 and Tyr150 , as well as the involvement of the drug molecule itself, in catalyzing the drug hydrolysis have remained debated. 35,36 In our previous study, 37 we were successful in unearthing the roles of active site and substrate residues during the acylation reaction. We found that the general base in the acylation is Lys67 , while the proton transfer to the β–lactam N occurs from Tyr150 via the drug carboxylate group or by a water molecule within the cavity of the active site.

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Interpretation of the full catalytic cycle of antibiotic resistance by CBL also requires the understanding of the mechanism of the deacylation step. Moreover, it is important to know how CBL carry out deacylation efficiently and reforms the active site. If a drug can be designed to have a very slow deacylation step, it will act as an inhibitor for CBL. This adds further interest in discerning the molecular level details of the hydrolysis of acyl–enzyme intermediate within CBL. Many experimental and theoretical studies have been devoted to gain insights into the deacylation mechanism of CBL based on which a number of hypotheses have been proposed. 38–45 A high resolution crystal structure of the enzyme bound to the transition state analogue of the deacylation reaction is in support of the existence of neutral Tyr150 during the deacylation reaction. 38 Based on this, Chen et al. disfavored any significant role of Tyr150 in the activation of the hydrolytic water molecule during deacylation. The existence of neutral Tyr150 in CBL was also supported by Ishiguro and coworkers based on their pKa measurement of Tyr150 . 39 However, their study was limited only to the apoenzyme. Several other studies carried out to explore the deacylation step in CBL proposed that deprotonated Tyr150 is the general base in the deacylation step. On determining the crystal structure of acyl– enzyme complex of CBL and lorcarbef drug molecule, Shoichet and coworkers suggested that Tyr150 in conjunction with the ring nitrogen of the substrate would stabilize the hydrolytic transition state. 40 They proposed that either the substrate or an anionic Tyr150 would play the role of the general base. Winkler and coworkers 41 recognized that the position occupied by Tyr150 at the active site of CBL is identical to that of the catalytic base residue, His57 , in trypsin. This observation led them to propose that Tyr150 is the catalytic base. Same interpretation was also made by other studies based on the supporting interactions noticed with the analogues of the tetrahedral intermediates such as phosphonate and boronic acid derivatives. 46–48 On the contrary, Mobashery and colleagues 42 proposed a substrate–assisted hydrolysis where lactam–nitrogen acts as the general base for the deacylation, in line with one of the proposals put forward by Shoichet and coworkers. 40

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A few computational studies have also reported the deacylation mechanism. Using static quantum mechanics/molecular mechanics (QM/MM) techniques, Gherman et al 43 studied the first step of deacylation, i.e. the activation of the catalytic water, in CBL. They proposed a concerted Tyr150 /Lys67 mechanism where the activation of the catalytic water by neutral Tyr150 occurs with a subsequent proton transfer from Tyr150 to Lys67 . The authors also reported that the electrostatic environments around Tyr150 , i.e. hydrogen bonding interactions between Tyr150 and Lys67 /Lys315 are essential to facilitate the hydrolysis in CBL. On the other hand, using a simplified QM model for the active site, Hata et al 44 proposed that Tyr150 serves as the catalytic base whereas Lys67 provides a proton to Ser64 , and the activation of catalytic water by Tyr150 governs the rate determining step. However, in these computational studies the dynamic aspects of solvated protein, in particular the active site residues, active site water molecule(s), and the drug molecule were not accounted for. Due to the flexible nature of the active site, protein and the solvent molecules, it is vital to obtain free energy estimate for the reaction barrier accounting the entropic contributions. Moreover, Gherman et al 43 studied only the formation of the tetrahedral intermediate, but not the subsequent protonation of Ser64 . Hata et. al. 44 computed only the potential energy barrier of the rate determining step, and is considerably higher than both the experimental estimate 49,50 and that computed by Gherman et al, 43 which is possibly due to the insufficient QM model and the non–dynamical approach followed. Thus, it becomes crucial to revisit the complete deacylation mechanism in CBL using molecular dynamics (MD) methods, by taking into consideration the fluctuations and dynamics of the fully solvated protein. Our previous studies have clearly demonstrated the dynamic nature of the protonation states and the active site structure along the extent of reaction. 37,51 It is vital to treat the active site residues and the drug molecule dynamically and quantum mechanically at the same time. In the present study, we have investigated the deacylation mechanism of CBL–cephalothin (see Figure 2 ) drug–enzyme complex , employing QM/MM 52 MD together with the metady-

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H N S

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1 7

O

8

O9

S

6

2 3

5

N

O

O

4

COO

CH3

Figure 2: Structure of cephalothin namics 53,54 approach, thus incorporating finite temperature effects and dynamics of the fully solvated protein while simulating the reaction. The combination of QM/MM and metadynamics techniques is ideal to study complex enzymatic reactions. 55 By combining the results of our previous study on the acylation mechanism of cephalothin, 37 we present here the complete mechanism and overall kinetics of cephalothin hydrolysis by CBL.

Methods and Models The starting structure of the acyl-complex of CBL and cephalothin was taken from the X–ray crystal structure corresponding to the PDB ID 1KVM. 56 Both Tyr150 and Lys67 were taken in their neutral state in the starting structure, in accordance with the final structure observed after the R2 group (i.e. acetoxy group) release in our previous study. 37 The protein was fully solvated with 12784 TIP3P water molecules in a periodic simulation box of dimensions 78 × 84 × 72 Å3 . 4 Na+ and 6 Cl− ions were also added to neutralize the whole system. The parm99 version of the AMBER force field 57 was used to define the potential of the protein structure while the potentials of cephalothin residue was expressed using the GAFF force field. RESP point charges for cephalothin and Ser64 were computed using the RED software 58 (see Supporting Information). A non-bonding interaction cutoff of 15.0 Å was chosen for all the simulations. Molecular mechanics (MM) calculations were carried out using the AMBER suite of programs. 59 6000 steps of geometry optimization was performed prior to the MM MD equilibration simulations. Solvent molecules together with the drug were relaxed, followed by the 6


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protein side–chain and the protein backbone in a step–wise manner. MM MD equilibration simulations were executed with a time step for 1 fs for integrating the equations of motion. At first, N P T ensemble equilibration was carried out (for about 1 ns) using a Berendsen barostat at 1 atm and Langevin thermostat at 300 K, till a satisfactory convergence was achieved in the (running average of the) density. Eventually a 5.5 ns N V T MD simulation was performed with the equilibrated density obtained from the N P T simulation. N V T MD simulations were performed till a reasonable convergence was observed in the RMSD of the protein backbone. MM MD simulation was followed by QM/MM MD simulation employing the CPMD/GROMOS interface code. 60 The side-chains of Ser64 , Lys67 , Tyr150 and Lys315 , and the whole cephalothin molecule together with the catalytic water were treated by QM. The capping H atoms were placed between the Cγ–Cδ bond of Lys67 and Lys315 and between the Cα–Cβ bond of Ser64 and Tyr150 . The capping atoms were constrained along the corresponding bonds connecting QM and MM regions. The QM system was described using plane wave density functional theory together with PBE exchange–correlation functional 61 and ultrasoft pseudopotentials. 62 The QM supercell size was 21×25×21 Å3 and a plane wave cutoff of 30 Ry was used for the plane–wave expansion of wavefunctions. The scheme proposed by Laio et al 63 was chosen for the QM-MM electrostatic coupling. Dynamics of the QM part was carried out using the Car-Parrinello MD 64 scheme. A fictitious mass of 700 au was assigned to all the orbital degrees of freedom. A time step of 0.145 fs was used for integrating the equations of motion in the QM/MM runs. The QM part and the rest of the system were thermostated using two different set of Nosé–Hoover chain thermostats at 300 K. 65 Extended Lagrangian metadynamics simulations 54,66,67 were performed to model the deacylation reaction. The technical details of the metadynamics simulation are given in the Supporting Information, and are identical to our previous study. 37 Free energy barrier for a reaction was computed by analyzing the reconstructed free energy surface, which in turn

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was obtained by summing the negative of the deposited biasing potentials in the course of a metadynamics simulation.

Results Acyl–Complex Structure of CBL and Cephalothin An MM MD simulation was first performed starting with the acyl–complex structure of CBL and cephalothin (structure EI). The maximum RMSD of the protein backbone measured in this simulation was less than 1.2 Å with respect to the X–ray structure of AmpC β-lactamase in complex with covalently bound cephalothin (1KVM) 56 (See Figure 3), providing confidence on the force–field and the simulation setup. Stable hydrogen bonding interactions were observed between Tyr150 and neighboring Lys67 and Lys315 , throughout the simulation (see Table 1). Two conformations of the active site, EIa and EIb, were distinguished with the major difference noticed in the interactions of Lys67 with Ala220 and Asn152 . Lys67 interacts with Ala220 and Asn152 in one configuration (EIa) whereas it interacts only with Asn152 in the other (EIb); see FigSI 2. The Lys67 · · · Ala220 interaction is a common feature seen in the X-ray structures of acyl-enzyme complex of CBL and the substrates. 56,68–72 Though both of the configurations were nearly equally populated and underwent rapid configurational exchange, EIb was found relatively more stable, during the 5.5 ns of N V T simulation (occupancies of structures EIb and EIa are 62 and 38 %, respectively) (see also Figure 3). However, the interactions noticed in EIa correlate well with the available X–ray crystal structures representing the acyl–enzyme complex of CBL and substrates, hence we concluded that EIa would be a good starting structure for the deacylation reaction. Also, we have shown later that the conversion between EIa and EIb was observed during the metadynamics simulation of hydrolysis reaction indicating that this choice of starting structure cannot change our prediction of reaction mechanism and free energies. Carbonyl group of the drug was well situated in the oxyanion hole created 8


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by Ser64 NH and Ala318 NH. Besides, a water molecule corresponding to the deacylating water (Wat), was found inside the active site during most of the simulation run. The structure after the 5.1 ns of classical MD simulation corresponding to the EIa (hereafter called as structure EI), was taken as the starting structure for the QM/MM simulations. Also, an additional structure EI’ was modeled from EI by transferring the proton of N5 to C4 carboxylate. This is to account for a proton transfer observed from N5 to C4 carboxylate in the last step of the acylation reaction concomitant with the release of the R2 group (as in Ref. 37 ). Two independent QM/MM simulations, of length 5 and 10 ps, were first performed starting with structures EI and EI’, respectively. Two wall potentials were applied along the distances between OWat and Tyr150 Oη , and between OWat and Cep362 C9 at 3.5 and 4.3 Å, respectively, to maintain the water within the active site close to the reaction center. An intermittent proton transfer was observed between Lys67 and Tyr150 during both these simulations, though the neutral Tyr150 was mostly seen in the trajectories (See FigSI 3 and FigSI 5). Interestingly, for the QM/MM simulations with the EI’ structure, we observed a proton transfer from C4 carboxylate group to N5 , resulting in the formation of EI (see FigSI 4). Hence, we conclude that EI’ is metastable and quickly transformed to EI. This also imply that metadynamics simulations of the deacylation reaction with the starting structures EI and EI’ would essentially lead to the same results.

Tetrahedral Intermediate (TI) Formation To model the formation of the tetrahedral intermediate, we started a metadynamics simulation starting from the structure EI’. The following collective variables were used to explore the deacylation reaction in CBL: (a) the coordination number of Tyr150 Oη to the protons of Wat (CV1), (b) the distance difference d[WatO-Cep362 C8 ] – d[Cep362 C8 -Ser64 Oγ ] (CV2), and (c) the coordination number of Ser64 Oγ to the terminal protons of Lys67 (CV3). CV1 was used to accelerate the proton transfer from Wat to Tyr150 . Since no stable interactions 9



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Figure 3: Backbone RMSD (blue color; left axis) and dihedral angle Cγ -Cδ -Cǫ –N of Lys67 , φ (black color; right axis) along the N V T simulation of acyl-complex of CBL and cephalothin with respect to the PDB structure ID: 1KVM. Dotted line is shown to differentiate the conformers of Lys67 , EIa and EIb (along the φ coordinate) characterized during the simulation.

Table 1: Average distance (in Å) between selected residues in the active site of equilibrium structure of acyl-complex of CBL and cephalothin computed from the MM MD simulation. Standard deviations are shown in brackets. Distance Tyr150 Oη · · · Lys67 Nζ

2.99 (0.40) Tyr150 Oη · · · Lys315 Nζ 3.16 (0.40) Lys67 Nζ · · · Asn152 Oδ 2.89 (0.14) Lys67 Nζ · · · Ala220 O (EIa) 3.14 (0.25) Lys67 Nζ · · · Ala220 O (EIb) 4.59 (0.33) CepO9 · · · Ser64 H 2.02 (0.15) CepO9 · · · Ala318 H 2.00 (0.14)

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of Wat with other potential basic residues like Lys67 , N5 and the carboxylate group of drug were observed during the MM and QM/MM MD simulations, these candidates are less likely to act as the base. For this reason, the possibility of Tyr150 acting as the base was the only one included in CV1. CV2 was chosen for sampling the nucleophilic attack of Wat onto the carbonyl carbon of cephalothin and the breaking of bond between Ser64 and cephalothin. CV3 was used for sampling the proton transfer from Lys67 to Ser64 . Similar to the QM/MM simulation, a wall potential was placed at 4.0 Å along the distance between Tyr150 Oη and OWat . A wall potential was also active at 2.65 Å along the CV2 to avoid sampling the structures irrelevant for the chemical reaction. With this setup, we were able to successfully simulate the nucleophilic attack of water. The reconstructed free energy surface from this simulation is shown in Figure 4. The molecular mechanism of nucleophilic attack was scrutinized by analyzing the trajectory of this simulation. As expected, a proton transfer from C4 carboxylate group to N5 resulting in EI was initially observed. The interaction between Lys315 and Tyr150 , which was maintained during the QM/MM simulation, was lost during the metadynamics simulation after the formation of EI. Following this, Lys315 was involved in the hydrogen bonding interactions with Tyr112 , a water molecule, and Glu272 (not shown in Figure 4). Lys67 interactions with Tyr150 , Asn152 and Ala220 were found stable throughout the reaction. During most of the simulation, the carbonyl group was situated in the oxyanion hole (oxyanion hole-1) formed by the backbone NH group of Ser64 and Ala318 . Interestingly, the N5 H5 group of the cephalothin along with the backbone NH group of Ala318 was found to act as an oxyanion hole (hereafter called oxyanion hole-2) during the reaction when oxyanion hole-1 was disturbed (see FigSI 7). The O9 carbonyl oxygen came out of the oxyanion hole-1 during the approach of Wat close to Cep362 C8 and fit into the oxyanion hole-2. Proton transfers were repeatedly observed between Tyr150 and Lys67 before the system crossed the barrier. Also, both configurations of Lys67 (EIa and EIb) were characterized during the metadynamics simulation. Subsequently, a proton transfer occurred from Wat to Tyr150 Oη , with

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CV3

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CV2 (A)

12 6 0

∆F (kcal/mol)

15 ∆F (kcal/mol)

EI’/EI TI

CV1

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TSEI-TI

6 5 0

TS EI−TI

TI

0 EI’/EI

(b)

(a) Tyr150 Cephalothin Wat Lys315 Lys6

7

Ser64 Ala318

EI’

TS EI−TI

TI

(c) Figure 4: (a) Reconstructed free energy surface, (b) free energy profiles and (c) snapshots, for the nucleophilic attack of water, first step in the deacylation reaction of CBL with cephalothin. The free energy surface, in (a), is visualized as three dimensional contour surfaces for various free energy isovalues.

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simultaneous movement of a proton from Tyr150 Oη to Lys67 Nζ (see Figure 5). Concomitantly the bond formation between OW1 and Cep362 C8 occurred, resulting in the formation of the tetrahedral intermediate structure, TI (see Figure 4). The free energy barrier of this process was computed to be 12 kcal mol−1 . In TI, Ser64 Oγ is in deprotonated state, but the complete dehydroxylation would require a proton transfer from Lys67 . However, this proton transfer was not seen in our metadynamics simulation, instead, the reverse reaction took place where Wat got detached from cephalothin by taking back its proton from Tyr150 . The free energy barrier of this reverse process was calculated to be 6 kcal mol−1 . The occurrence of reverse reaction also indicates that the barrier for the proton transfer to Ser64 is higher than that for the TI→EI reaction.

Figure 5: Distances WatO to Cep362 C8 (blue), Tyr150 Oη to WatH (red) and Tyr150 Oη to Tyr150 Hη (black) during the nucleophilic attack of Wat to carbonyl carbon of cephalothin.

Mechanism of Protonation of Ser64 Oγ To explore the second elementary process in the deacylation reaction, i.e. the detachment of Ser64 from the drug and the subsequent protonation of Ser64 Oγ , a metadynamics simulation was performed starting with the tetrahedral intermediate structure TI obtained after the previous metadynamics simulation. A 5 ps of QM/MM simulation was first performed starting with TI. A proton transfer from Cep362 O9 to Cep362 N5 was observed several times 13



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during this simulation (see FigSI 6). The proton transfer to carbonyl O9 was not surprising as it carries a partial negative charge in the tetrahedral intermediate structure TI (see Figure 6c). The following CVs were used for the metadynamics simulation (a) the coordination number of Ser64 Oγ to the terminal protons of Lys67 (CV3), (b) the distance between Ser64 Oγ and Cep362 C8 (CV4), and (c) the coordination number difference C [Cep362 N5 · · · H5 ] - C [Cep362 O9 · · · H5 ] (CV5). The importance of CV3 is already discussed before. CV4 was selected to sample the distance between Ser64 Oγ and Cep362 C8 ; this is required for the detachment of Ser64 from the drug. CV5 was used to sample the proton transfer between Cep362 N5 and Cep362 O9 . A wall potential was applied on the distance d[Cep362 C8 · · · WatO] at 1.85 Å to avoid TI to cross back to the minimum EI. Using these coordinates, we were able to simulate the cleavage of covalent bond between of Ser64 and the drug molecule and, proton transfer to Ser64 Oγ . The reconstructed free energy surface is shown in Figure 6. Three minima can be observed in this reconstructed free energy surface. A proton transfer from Cep362 N5 to Cep362 O9 resulted in an intermediate TI’. The free energy barrier for this proton transfer process is about 3 kcal mol−1 , while the reverse barrier (TI’→TI) is about 2 kcal mol−1 . Thus the tetrahedral intermediate TI has a dynamic structure at equilibrium, where H5 is delocalized between the Cep362 O9 and Cep362 N5 . The carbonyl group was largely bound to the oxyanion hole-2 during this simulation. Lys67 , in its protonated form, interacts mainly with neutral Tyr150 in structure TI. Eventually a proton transfer from Lys67 to Ser64 Oγ , with the subsequent detachment of Ser64 from cephalothin, was observed. This also resulted in breaking of interaction between Tyr150 and Lys67 (see Figure 7). Subsequently, Tyr150 lost its contacts with the hydrolyzing Wat, but formed hydrogen bonds with both Lys315 and another water molecule that has diffused into the active site during this reaction (see Figure 6). This event completes the hydrolysis of cephalothin. The N5 was protonated while converting from TI to EP (see Figure 6). The overall barrier for TI→EP is 8 kcal mol−1 . Note that TI’ was not leading to the hydrolyzed product.

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CV5

CV3

∆F (kcal/mol)

12

3

0

6

∆F (kcal/mol)

EP

8

CV4 (A)

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TI’ TI

1 0

8

8

3

4 0

> k2 , thus

kcat =

k 2 k3 ≈ k2 k 2 + k3

Thus, the overall free energy barrier for the reaction is about 17 kcal mol−1 , which agrees very well with the experimental estimates of 15–17 kcal mol−1 . 33,34 The kinetics of drug hydrolysis is dependent on the drug and the enzyme. Only a few studies have also been devoted to understand the rate-limiting step in CBLs and none of these studies have explicitly considered cephalothin, to our knowledge. Some previous studies calculated k2 is higher than k3 for penicillin and cephalosporins 50,80,81 and suggested that deacylation must be the rate-limiting step for these drugs in CBL. Considering the inconsistency noticed in the previously reported

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2 2 I1 ∆ F (kcal/mol)

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17

>19

>2 6

7 ES I2

>6

8

EP

12

4

7

I3

~1 EI’

TI ~1 EI

Figure 9: Free energy profile for the cephalothin hydrolysis catalyzed by CBL. Here ES represents the Henry-Michaelis complex of CBL and cephalothin whereas I1, I2 and I3 denote different intermediates observed during the acylation of cephalothin. Barrier for the acylation steps are taken from Ref. 37 kinetic analyses, for example the change in the kcat and kcat /Km values for cephalothin on Lys-67-Arg mutant enzymes in different studies were found highly contrasting, 33,82 we believe that more experimental studies are required to validate our finding. Finally, it is noted in passing that the observation of facile proton transfer within the active site could be due to the underestimation of the proton transfer barrier by the PBE functional. Based on the recent benchmark studies 83,84 conducted on various systems, PBE and other GGA functionals show an average error of about 4 kcal mol−1 . The barrier for proton transfer between Lys67 and Tyr150 in CBL–apoenzyme was found to underestimate by about 1 kcal mol−1 with the PBE functional in our earlier study. 51 Thus, we may conclude that proton could be more localized to Tyr150 in the acyl–enzyme complex than what we have observed with the PBE functional. The crucial steps in the hydrolysis reactions studied here have spontaneous proton transfers occurring after a C–O or a C–N bond formation or dissociation, and thus we do not expect large discrepancy in the overall mechanism and kinetics. The reaction barriers of cephalothin acylation reaction in CBL were also mostly found to be underestimated (up to 2 kcal mol−1 ) by PBE. 37

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Conclusions Through extensive QM/MM metadynamics simulations, we explored the deacylation reaction of CBL–cephalothin acyl–enzyme complex. Tyr150 is largely in the protonated form in the acyl–enzyme complex, however, proton transfer from Tyr150 to Lys67 occurs prior to the hydrolysis, converting the former to a basic residue. Further, breakage of the hydrogen bonding interaction between Tyr150 and Lys315 allows Tyr150 to align towards the catalytic water, while maintaining the hydrogen bonding interaction with Lys67 . A favorable orientation of the drug toward the attacking water molecule also takes place by shifting the carbonyl group of the drug from oxyanion hole–1 to oxyanion hole–2. The nucleophilic attack of water to the carbonyl carbon atom of the acyl–complex occurs in tandem with the proton transfer of the attacking water molecule to Tyr150 . In a subsequent step, the cleavage of the acyl bond takes place with simultaneous proton transfer from Lys67 to Ser64 . The hydrogen bonding interaction between Tyr150 and Lys315 is reformed after the complete reaction. For the deacylation reaction, Tyr150 acts as the general base and Lys67 is the proton donor. It is interesting to note that Tyr150 and Lys67 interchange their catalytic roles in the deacylation step compared to the acylation step. For the cephalothin hydrolysis, we predict that the rate of the deacylation reaction is higher than the rate of the acylation reaction, and kcat ≈ k2 . Thus acylation reaction is the rate–determining step in the cephalothin hydrolysis by CBL. The apparent free energy barrier for the cephalothin hydrolysis is computed to be 17 kcal mol−1 , which is in good agreement with the experimental kinetic data. It is now evident that deprotonation of Tyr150 by transferring its proton to Lys67 is a critical step (although not the rate–limiting) occurring prior to the activation of the catalytic water molecule. Proton transfer between these residues is facilitated by the proximity of these residues within the active site. Thus, inhibitor design may target to disfavor the hydrogen– bonding interaction between these residues and thus maintaining the protonated form of Tyr150 in the acyl–enzyme complex, thereby increasing free energy barrier and slow down 21



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the deacylation reaction.

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Acknowledgement Authors thank IIT Kanpur for providing the HPC facility. RT thanks CSIR, India for his Ph.D. fellowship. Authors gratefully acknowledge the funding by the Department of Biotechnology (DBT).

Supporting Information Available Supporting Information has analysis of the trajectories from molecular dynamics simulations and technical details of the metadynamics simulation setup. Full citation for references 11 and 59 are provided. Charges of Ser64 and cephalothin residues are available as a text file. Structures of intermediates and transition states are deposited as .pdb files. This material is available free of charge via the Internet at http://pubs.acs.org/.

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