MM Simulation Study of Intramolecular Proton Transfer in

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A Hybrid QM/MM Simulation Study of Intramolecular Proton Transfer in the Pyridoxal 5'-Phosphate in the Active site of Transaminas: Influence of Active Site Interaction on Proton Transfer Sindrila Dutta Banik, and Amalendu Chandra J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp506196m • Publication Date (Web): 27 Aug 2014 Downloaded from http://pubs.acs.org on September 11, 2014

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The Journal of Physical Chemistry

A Hybrid QM/MM Simulation Study of Intramolecular Proton 0 Transfer in the Pyridoxal 5 -Phosphate in the Active site of Transaminas: Influence of Active Site Interaction on Proton Transfer

Sindrila Dutta Banik and Amalendu Chandra∗ Department of Chemistry, Indian Institute of Technology, Kanpur, India 208016.

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Abstract 0

Pyridoxal 5 -Phosphate (PLP) Schiff base, a versatile cofactor, exhibits a tautomeric equilibrium which involves an intramolecular proton transfer between N-protonated zwitterionic ketoenamine tautomer and O-protonated covalent enolimine tautomer. It has been postulated that for the catalytic activity, the PLP has to be in zwitterionic ketoenamine tautomeric form. However, the exact position of the tautomeric equilibrium of Schiff base in the active site of PLP-dependent enzyme is not known yet. In the present work, we investigated the tautomeric equilibrium for the external aldimine state of PLP Aspartate (PLP Asp) Schiff base in the active site of Aspartate aminotransferase (AspAT) using combined quantum mechanical and molecular mechanical (QM/MM) simulations. The main focus of the present study is to analyze the factors which control the tautomeric equilibrium in the active sites of various PLP dependent enzymes. The results show that the ketoenamine tautomer is more preferred than the enolimine tautomer both in the gas and aqueous phases as well as in the active site of AspAT. Current simulations show that the active site of AspAT is more suitable for ketoenamine tautomer compared to the enolimine tautomer. Interestingly, the Tyr225 acts as a proton donor to the phenolic oxygen in ketoenamine tautomer while, in the covalent enolimine tautomer, it acts as a proton acceptor to the phenolic oxygen. Finally, the metadynamics study confirms this result. The calculated free energy barrier is about 7.5 kcal/mol. A comparative analysis of the microenvironment created by the active site residues of three different PLP dependent enzymes (Aspartate aminotransferase, Dopa decarboxylase and Ala-racemase) has been carried out to understand the controlling factor(s) of the tautomeric equilibrium. The analysis shows that the intermolecular hydrogen bonding between active site residues and the phenolic oxygen of PLP shifts the tautomeric equilibrium towards the N-protonated ketoenamine tautomeric form.

Keywords: Keto-enol tautomerism, Transaminase, PLP dependent enzyme, QM/MM Simulation


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1

Introduction 0

Pyridoxal 5 -Phosphate (PLP) is a versatile cofactor which catalyzes a variety of chemical transformations, such as racemization, decarboxylation, transamination reactions etc 1–4 . A group of enzymes uses PLP as a cofactor during catalysis to reduce the energy barrier 5–8 . According to the crystallographic study, 5–9 initially the PLP forms a covalent bond with a conserved lysine residue of PLP dependent enzyme which is usually referred to as the internal aldimine state. Subsequently, the PLP-enzyme complex reacts with the substrate and the bond between the lysine of active site and PLP is broken. As a result of this reaction, a new Schiff base is formed between the PLP and substrate amino acid which is usually referred to as the external aldimine state. This step is commonly known as the transimination reaction which is a prerequisite for functionality of all PLP dependent enzymes 10,11 .

The PLP contains four sites such as imino group with the corresponding imino nitrogen referred to as N, phenolic group with the corresponding phenolic oxygen referred to as O3, pyridine group with the corresponding pyridine nitrogen referred to as N1 as well as a phosphate group (PO4 ) 12 . This is shown in Figure 1. The protonation state of imino nitrogen, phenolic oxygen and pyridine nitrogen depends on the surrounding environment. The pyridine nitrogen (N1) of the external aldimine has a pKa of about 5.8 in aqueous solution and remains as unprotonated under physiological condition 13,14 . The protonation state of the imino nitrogen (N) and phenolic oxygen (O3) are mutually dependent. A proton forms an intramolecular hydrogen bond (shown as O–H–N) between the imino nitrogen and phenolic oxygen of PLP. It can undergo an intramolecular proton transfer from N-protonated ketoenamine (zwitterionic) Schiff base to O-protonated enolimine (covalent) Schiff base, giving rise to a tautomeric equilibrium as shown in Figure 2. This equilibrium is an important factor that affects the reactivity of the PLP Schiff base in the enzyme active site 1,14 . Therefore, it is important to know the exact position of the proton between imino nitrogen and phenolic oxygen of PLP Schiff base in order to understand the reactivity of the enzyme. In the present paper, we studied the intramolecular proton transfer reaction for the external aldimine state of PLP-Asp Schiff base in the active site of Aspartate aminotransferase (AspAT) using combined quantum mechanical-classical mechanical (QM/MM) method to elucidate how the enzyme active 3 Environment ACS Paragon Plus


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site affects the tautomeric equilibrium.

AspAT is a PLP dependent enzyme which reversibly catalyzes the conversion of amino acid into keto acid as well as keto acid to amino acid via ping-pong bi-bi mechanism 15–18 . The X-ray crystal structure shows that the apo-enzyme (absence of PLP) is catalytically inactive 19–22 . In the resting state, the PLP is covalently linked with Lys258 (internal aldimine state). This internal Schiff base is converted into external PLP-Asp Schiff base via displacing Lys258 by substrate Asp 23–30 . The PLP is positioned at the bottom of the active site pocket and the active site residues interacts with the external aldimine state of PLP-Asp Schiff base via hydrogen bonding interaction, electrostatic interaction, hydrophobic interaction 15 . This is shown in Figure 3. The phosphate moiety of PLP contains two negative charges and interacts with positively charged Arg266. It also forms hydrogen bonds with hydrogen bond donors such as Gly108, Thr109, Ser255, Tyr70* (* indicates the residue of another subunit). The pyridine ring of PLP interacts with Ala224 on one side and on the other side with indole ring of Trp140. The active site residue Asp222 forms a salt bridge with the pyridine nitrogen of PLP. The phenolic oxygen of PLP forms hydrogen bonds with Asn194 and Tyr225 and is expected to play a significant role in the tautomeric equilibrium. Several experimental 13,31–41 as well as computational studies 39,41–47 have been carried out to rationalize the tautomeric equilibrium and the nature of the intramolecular hydrogen bond between the phenolic oxygen and imino nitrogen as well as intermolecular hydrogen bond between pyridine nitrogen and surrounding groups in the PLP Schiff base. Model studies have shown that the Schiff base compounds exhibit a keto-enol tautomerism between the N-protonated ketoenamine (zwitterionic) form and O-protonated enolimine (covalent) form. Two forms interconvert very rapidly via intramolecular proton transfer. However, the tautomeric equilibrium is influenced by various factors such as the solvent polarity 13,38,39 , substituent on the imino group, local polarity around the intramolecular hydrogen bond as well as protonation state of the pyridine ring 40,41 . It was shown that the ketoenamine form of the Schiff base is preferred with increasing polarity of an aprotic solvent 38 . Solid state NMR study of model compounds has shown that the intra- and intermolecular hydrogen bonds exhibit a cooperative coupling where the protonation of the pyridine nitrogen prefers the ketoenamine form of the Schiff base 40 . This 4 Environment ACS Paragon Plus

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coupling depends on the substituent on imino group as well as local polarity around the intramolecular hydrogen bond. If the substituent is an aliphatic group (such as internal or external aldimine) then the Schiff base exhibits this coupling while this coupling is lost if the substituent is an aromatic group 40 . Similarly, the coupling is also lost in aqueous solution but preserved in methanol 39 .

Earlier model studies have shown that the protonation state of PLP Schiff base is greatly influenced by the local polarity 39,41 . It also has been postulated that for the proper catalytic activity of the PLP dependent enzyme, the PLP Schiff base, should be in N-protonated ketoenamine state. A positive charge over imino nitrogen of PLP Schiff base is a prerequisite 1,2 for function of the enzyme. For this purpose, the bridging proton of the intramolecular (O–H–N) hydrogen bond has to be transferred to imino nitrogen from phenolic oxygen. That is the tautomeric equilibrium has to shift towards the ketoenamine conformation. Since the protonation state of PLP strongly depends on the environment, it is important to investigate which kind of environment is realized by PLP in the active site of various PLP dependent enzymes. Also how the protonation state of PLP Schiff base is influenced by the microenvironment created by the enzyme is an issue that is poorly understood and remain to be investigate.

Although a wealth of information is available for the tautomeric equilibrium of PLP or PLP analogue Schiff base, the exact location of the bridging proton of PLP in the enzyme active site is yet to be known. A recent study has shown that in the active site of Dopa decarboxylase (DDC), the enolimine conformation is preferred over ketoenamine form 14 . However, the exact position of the bridging proton of PLP in the active site of AspAT remains unanswered. The model study has revealed that the tautomeric equilibrium of PLP Schiff base depends on the electrostatic environment. The electrostatic environment created by an enzyme active site can be very different from that of another. So, it is expected that the position of the tautomeric equilibrium in AspAT may not be similar to what is found for the DDC. The goal of the present work is to understand the position of the tautomeric equilibrium in the active site of AspAT using computational methods.



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A related question is that which factors control the position of the tautomeric equilibrium in the enzyme active site. A first principle study has shown that protonation of the pyridine ring shifts the tautomeric equilibrium of PLP Schiff base towards the ketoenamine form for the model compound 42 . However, in the active site of DDC, the pyridine nitrogen of PLP Schiff base is protonated and the enolimine tautomer is preferred over ketoenamine tautomer 14 . While in case of Ala-racemase (AlaR) the pyridine nitrogen of PLP Schiff base is unprotonated and the enolimine tautomer is preferred 47 . So, the tautomeric equilibrium in the enzyme active site not only depends on the protonation state of the pyridine ring, there can be other factor too which control the equilibrium.

In the present study, first we investigated the tautomeric equilibrium of the external aldimine state of PLP-Asp Schiff base in the active site of AspAT. We further analyzed the factors which influence the equilibrium in the active site of PLP dependent enzyme. To study the tautomeric equilibrium, first we analyzed the energetics of the external PLPAsp Schiff base using static quantum-chemical approach. We further investigated the proton affinity values. Two independent empirical force field MD simulations have been performed for the N-protonated ketoenamine (zwitterionic) Schiff base and O-protonated enolimine (covalent) Schiff base which mutually differ in the protonation state of the substrate. Finally, we explored the free energy path of proton transfer process in going from N-protonated ketoenamine to O-protonated enolimine Schiff base employing metadynamics technique within QM/MM setup. A comparative analysis of the hydrogen bonding states between active site residues as well as substrates is carried out for AspAT, DDC and AlaR to explore the factors which control the tautomeric equilibrium.

The details of the calculations are described in section II. The results are discussed in section III and our concluding remarks are presented in section IV.

2

Details of simulations The X-ray crystal structure of AspAT (1ARG.pdb) 48 from E. Coli complexed with 0

PPD (pyridoxyl-aspartic acid 5 monophosphate) was used as the initial structure for the present calculations. The enzyme is a homodimer which consists of 396 amino acid residues in each subunit. Each subunit is comprised by two domains: one large domain 6 Environment ACS Paragon Plus

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and one small domain. There are two active sites and two PLPs per dimer. The two active sites are located at the interface of two subunits.

Crystallographic studies have revealed that in internal aldimine state the pyridine nitrogen of PLP-Asp Schiff base forms a strong OHN intermolecular hydrogen bond with the side chain carboxylic acid group of Asp222 32,33 . In the crystal structure, the O...N separation is 2.6 ˚ A . The N-H and O...H distances are estimated using NMR study as 1.09 ˚ A and 1.54 ˚ A respectively 33 . The pKa of pyridine nitrogen of external aldimine is 5.8 in water which is more basic than a carboxylic group. An NMR study also indicates that in the active site, the proton should remain on the pyridine nitrogen 32,33 . So in the present calculations, we kept the pyridine nitrogen of external PLP-Asp Schiff base as protonated. To study the intramolecular proton transfer reaction in external PLP-Asp Schiff base, we first built the structure of zwitterionic ketoenamine tautomer and then the covalent enolimine tautomer. The substrate (PLP-Asp Schiff base) was generated by modifying the structure of PPD. The phosphate group of PLP contains two negative charges and each of the two carboxylate groups contains a negative charges each. The protonation states of the ionizable residues were assigned corresponding to pH 7. Thus, Asp and Glu residues treated as anionic and Lys (except Lys258) and Arg as cationic. The active site residue Lys258, which is released from the internal aldimine Schiff base, was set neutral. The histidines are taken as neutral residues.

We first investigated the energetics of the external PLP-Asp Schiff base molecule using static quantum-chemical approach. The free energy difference (∆G) for the tautomeric equilibrium between the ketoenamine and enolimine tautomers was calculated for external PLP-Asp Schiff base in the gas phase (∆Ggas ), in presence of solvent (∆GP CM ) and in the active site of AspAT (∆Gactivesite ). To calculate ∆Ggas as well as ∆GP CM , the geometries were optimized at HF/6-311+G** level and the single point calculations were performed at B3LYP/6-311+G** level. While to calculate ∆Gactivesite , optimization has performed using ONIOM (HF/6-311+G**:PM3) level of calculation and corresponding single point energies were calculated at ONIOM (B3LYP/6-311+G**:PM3) method. In order to assess the effects of polar solvent on the value of ∆G for the tautomeric equilibrium, we have employed the polarizable continuum model (PCM) 49 . To calculate the ∆Gactivesite , a small model of active site was generated using the crystal structure. In 7 Environment ACS Paragon Plus


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addition to the external PLP-Asp Schiff base the model of active site includes Asn194, Asp222, Tyr225, Lys258, Arg266, Arg386, Arg292* (the numbering scheme corresponding to the scheme as in AspAT of E. Coli; PDB code: 1ARG). As these residues are in close proximity to the reactant in the active site and interact with the substrate, they are considered in the present model. The HF/6-311+G** level of calculations were performed for the substrate external PLP-Asp Schiff base while PM3 level of calculations were performed for the surrounding active site residues. The results are shown in Table I.

We further calculated the proton affinity (PA) for the two tautomers of PLP-Asp Schiff base (in external aldimine state) as the change in enthalpy associated with the addition of a proton to a molecule B in the reaction, B + H+ → BH+ . The proton affinity is calculated as PA = – ∆Eel – ∆(ZPVE) where, ∆Eel (= E(BH+ ) – E(B)) corresponds to the differences in ground state electronic energies of the protonated and unprotonated forms of base (B). ∆(ZPVE) is the difference in zero point vibrational energies of the protonated and unprotonated forms. The above equation assumes the PA to be dominated by energetic rather than pressurevolume effects 50,51 . In the present calculations, the ’base’ or proton acceptor, (B) corresponds to the imino nitrogen (N) for the zwitterionic ketoenamine tautomer and phenolic oxygen (O3) for the covalent enolimine tautomer. First, the gas phase proton affinity was determined. The gas phase properties provide information about the intrinsic behavior of a compound in absence of solvent and enzyme effects. Then, subsequently, we determined the proton affinity in presence of solvent and enzyme active site. PCM model was used to calculate the PA in solvent, and the model of active site, as described in the previous paragraph, was used to calculate the PA in enzyme active site. The results are shown in Table II. All the ab initio and DFT calculations were done using the Gaussian 09 program 52 .

To perform the molecular dynamics simulations, the enzyme system described above was solvated in a rectangular box of dimension 112 X 119 X 91 ˚ A3 containing 29833 8 Environment ACS Paragon Plus

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TIP3P water molecules. The crystal structure contains 500 water molecules and these crystallographic waters are also considered in the simulation. The resulting system has a net charge -22 and twenty two sodium ions were added to achieve the electrical neutrality. The final model of the enzyme system consists of 1,03,271 number of atoms.

All water molecules were first energy minimized for 500 steps while rest of the system was held constrained. This was followed by 500 steps energy minimization of the overall system. Subsequently the system was followed by 250 ps isothermal-isobaric (NPT) simulation with positional restraints to relax water, substrate and side chain of the amino acids gradually. Then the system was followed by 500 ps NPT simulation without positional restraints. After the initial setup, canonical (NVT) simulation was carried out for a time period of 2.5 ns. The generalized amber force field (GAFF) 53 was employed together with the RESP point charges (computed using RED package) 54 to describe the substrate while the amber (leaprc.ff99SB) force field 55 was used for the protein.

During the simulations, the temperature was maintained at 300 K using Langevin dynamics with a collision frequency of 1.0 ps−1 and pressure was maintained isotropically at 1 atm with relaxation time of 2 ps. The simulations were carried out with a time step of 1 fs. Covalent bonds involving the hydrogen atoms were constrained by the SHAKE algorithm. The particle-mesh Ewald method (PME) was employed to treat the long-range electrostatic interactions. The nonbonded cutoff was set to 10.0 ˚ A. Selected intramolecular as well as intermolecular separations are listed in Table III and Table IV, respectively.

The time scale of a chemical reaction depends exponentially on the free energy barrier. For example, a process which involves a free energy barrier of ∼ 0.15 kcal/mol can be easily observed by few picoseconds of QM/MM simulation 56 . On the other hand, a few nanosecond simulation has to be performed to observe an event that proceeds by crossing a barrier of 5 kcal/mol at 300 K 57 . Unfortunately, only a few tens of picoseconds can be accessed by QM/MM molecular dynamics simulations. So, to accelerate the ’rare event’, we employed the metadynamics technique 58–62 . In metadynamics method, the sampling is facilitated by the introduction of a small repulsive Gaussian potential (history dependent) to the Hamiltonian of the system. This potential acts on a selected number of degrees of freedom. These selected degrees of freedom often referred as the collective 9 Environment ACS Paragon Plus


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variable (CV). The choice of collective variable is crucial for the reliability of this method. In recent years, the metadynamics technique has been applied in various kinds of chemical and biological problems 63–72 .

Here, the classically equilibrated system of zwitterionic ketoenamine tautomer was subjected to hybrid QM/MM metadynamics simulation. The system was partitioned into quantum and classical parts as required for the QM/MM methods. All the atoms of the substrate external PLP-Asp Schiff base and the side chain of Lys258 were treated quantum mechanically. This part contained 52 atoms. The QM supercell used in the present case was 22 × 22 × 22 ˚ A3 . The QM/MM boundary was set between Cα -Cβ bond of Lys258. The dangling bond of Cβ atom in QM part was saturated by a capping hydrogen atom. The QM part was calculated using the Car-Parrinello molecular dynamics method while the MM part was treated using the GROMOS96 force field. The calculations were done using the CPMD code 73 .

We employed the BLYP exchange-correlation functional in the present calculations of the QM part. The core electrons of all the atoms of the QM part were treated by Troullier-Martins norm-conserving pseudopotentials and the plane wave expansion of the valence-electron wave functions was truncated at a kinetic energy of 70 Ry. The simulations were carried out with a time step 4 au and a fictitious electronic orbital mass of 400 au. In order to reduce the importance of quantum effects, all hydrogen atoms were given the mass of deuterium. As described above, the initial configuration was obtained from classical molecular dynamics simulation. Subsequently, before metadynamics simulations, we equilibrated the system for about 5 ps in an NVT ensemble at 300 K using the Nose-Hoover chain method.

The metadynamics technique allows the system to find out the minimal energy path through the action of collective variables as well as provides a free energy landscape. For the present calculations, the biasing potentials used were normal Gaussian functions. The hill width and height was fixed at 0.04 and 0.0005 au (0.313 kCal/mol), respectively. In the present study, we used coordination number c[A-B] as CVs 61 which measures the total


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coordination number between two sets of atoms A and B, defined as c(A − B) =

X

0 )6 1 − (RAI /RAB 0 )12 1 − (RAI /RAB

(1)

Further details of CVs employed in the present work will be discussed in the result section.

3

Result and Discussion We have studied the intramolecular proton transfer reaction of external aldimine

state of PLP-Asp Schiff base in the active site of AspAT using the combined QM/MM method. For this, first we investigated the energetics of the external PLP-Asp Schiff base molecule using static quantum-chemical approach. We further investigated the proton affinity values. We performed two independent empirical force field MD simulations for the N-protonated ketoenamine (zwitterionic) Schiff base and O-protonated enolimine (covalent) Schiff base, which mutually differ in the protonation state of the substrate. Finally, we explored the proton transfer free energy pathway in going from N-protonated ketoenamine to O-protonated enolimine Schiff base by employing the metadynamics technique within QM/MM setup.

3.1

Quantum-chemical calculations We first investigated the energetics of the PLP-Asp Schiff base molecule in external

aldimine state using the B3LYP/6-311+G**//HF/6-311+G** level of calculations in the gas phase and in solvent. We also looked at the energetics of the external aldimine state of PLP-Asp Schiff base in the model of active site through B3LYP/6-311+G**:PM3//HF/6311+G**:PM3 level of calculations. The results are shown in Table I. The positive values of ∆G both in gas phase, in presence of solvent as well as in the active site of AspAT means that the N-protonated ketoenamine (zwitterionic) of external aldimine state of PLP-Asp Schiff base is more stable than the O-protonated enolimine (covalent) PLP-Asp Schiff base all cases. The energy difference between N-protonated ketoenamine (zwitterionic) Schiff base and O-protonated enolimine (covalent) Schiff base is 8.64 kcal/mol in the gas phase, 8.75 kcal/mol in aqueous medium and 6.91 kcal/mol in the small model of active site of AspAT. The significantly higher energy of the enolimine form in the active site of AspAT is due to unfavorable interaction between the substrate and active site residues. The results reflect that the ketoenamine tautomer of PLP-Asp Schiff base in 11 Environment ACS Paragon Plus


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external aldimine state would be preferred over enolimine tautomer in the active site of AspAT. The pyridine nitrogen is protonated, consequently the favorable electrostatic interaction between positive charge on the pyridine ring and negative charge of phenolic oxygen stabilizes the zwitterionic tautomer rather than neutral tautomer. The result is consistent with recent experimental 13 as well as computational studies 14 .

We further analyzed the proton affinity (PA) values for the phenolic oxygen (O3) of enolimine tautomer and imino nitrogen (N) of ketoenamine tautomer of external aldimine state of PLP-Asp Schiff base in gas phase, in presence of solvent as well as in the active site of AspAT using B3LYP/6-311+G**//HF/6-311+G** level of calculations. The results have been summarized in Table II. The results show that the PA of ketoenamine tautomer of external PLP-Asp Schiff base is greater than the enolimine tautomer both in the gas phase, in presence of solvent as well as in the active site of AspAT. The PA is maximum in gas phase followed by active site of enzyme and in presence of solvent for both tautomers. Similar trend is also observed for the side chain of amino acids 50,51 . The relatively higher value of PA for the ketoenamine tautomer suggests that the imino nitrogen (N) has greater tendency to take up a proton compared to the phenolic oxygen (O3) of enolimine tautomer. Alternatively, the tautomeric equilibrium will prefer to shift towards the ketoenamine tautomer direction.

3.2

Force Field MD Simulations We performed two independent empirical force field MD simulations for solvated

protein with ketoenamine tautomer and enolimine tautomer of external aldimine state of PLP-Asp Schiff base as substrate, respectively. The two tautomers mutually differ in their protonation state. The root mean square deviations (RMSD) of the protein backbone with respect to the first frame of the NVT simulation are shown in Fig. 4 for both systems. The RMSD values remain less than 2 ˚ A which means that the protein structure was stable throughout the 2.5 ns NVT simulation. The average separations (based on 2.5 ns NVT simulation) between the phenolic oxygen (O3) and imino nitrogen (N) (rO3−N ); phenolic oxygen (O3) and proton (rO3−H ); and imino nitrogen (N) and proton (rN −H ) as well as the dihedral angle φN −C4A−C4−C3 are listed in Table III. This dihedral angle signifies the planarity of the Schiff base. The average donor acceptor separation (rO3−N ) is ∼ 2.6 ˚ A for


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the two tautomers. The average dihedral angle value is also close to zero which reveals that the Schiff base is almost planar in both configurations. Form these results we can conclude that both tautomers form strong intramolecular hydrogen bonds.

The active site of AspAT where the PLP-Asp Schiff base is embedded is located at the subunit interface and constructed by two subunits. The PLP-Asp Schiff base is enclosed in a network of hydrogen bonding interactions coupled with hydrophobic interactions. It is already mentioned in the Introduction that these hydrogen bonding interactions and the surrounding environment guide the protonation state as well as the tautomeric equilibrium of the Schiff base. These two factors are crucial for controlling the reactivity of the cofactor. In the active site pocket of the enzyme, the active site residues are responsible for creating the hydrogen bonding interactions and also the surrounding environment.

The pyridine nitrogen (N1) of PLP-Asp Schiff base is protonated in the active site of AspAT and forms an ion pair interaction with active site residue Asp222 in both tautomers. This is shown in Figs. 5 and 6. The average separation between the H1 atom of PLP Schiff base and carboxylic oxygen atom of Asp222 is ∼ 2 ˚ A for both tautomers. This is shown in Table IV. The results show a strong hydrogen bonding between PLP and Asp222. The side chain of Asp222 contains negatively charged carboxylic acid group and stabilizes the protonated pyridine ring via electrostatic as well as hydrogen bonding interactions. The protonated pyridine ring acts as an electron sink to stabilize the carbanion intermediate which is crucial for the catalysis. It was also shown that the protonation state of pyridine nitrogen has an influence on the reaction path 74 . So, in the active site of AspAT, the protonation state of pyridine is guided by Asp222 and the proper position and orientation of the same is controlled by secondary interactions between Asp222 and His143 as well as between Asp222 and surrounding waters. These secondary interactions play important role in stabilizing as well as maintaining the protonation state of the pyridine nitrogen (N1) of external PLP-Asp Schiff base. Notably, the O...N separation obtained from the present calculation is greater than the O...N separation observed in the crystal structure in internal aldimine state 32 . This result indicates that the pyridine-Asp222 hydrogen bond is weaker in external aldimine state relative to the internal aldimine state.



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Similar to the protonation state of the pyridine ring, the tautomeric equilibrium between ketoenamine and enolimine conformation of PLP is controlled by the hydrogen bonding interactions as well as electrostatic interactions between PLP Schiff base and active site residues. For the AspAT, a Tyr residue (Tyr225) as well as an Asn residue (Asn194) are in close proximity to the phenolic oxygen (O3) of PLP-Asp Schiff base which can form hydrogen bonds with the phenolic oxygen (O3). The present results show that the Asn194 acts as a hydrogen bond donor for both the ketoenamine tautomer as well as the enolimine tautomer (shown in Figs 5 and 6). The amide group of Asn194 forms two hydrogen bonds with PLP-Asp Schiff base; one with O3 atom and the other one with oxygen atom of the carboxylic group of PLP. The average separation between O3 atom of PLP and hydrogen atom of the amide group of Asn194 is 1.85 ˚ A in ketoenamine tautomer while the distance is 2.05 ˚ A in that enolimine tautomer as shown in Table IV. The results show that the Asn194 forms relatively strong hydrogen bonds with ketoenamine tautomer compared to that with the enolimine tautomer of the PLP Schiff base.

Interestingly, the Tyr225 behaves differently with two tautomers. It acts as hydrogen bond donor for the ketoenamine tautomer while, in case of the enolimine tautomer, it acts as a hydrogen bond acceptor. In the zwitterionic ketoenamine tautomer, where the O3 atom is negatively charged, the phenolic hydrogen of the side chain of Tyr225 forms a hydrogen bond with the O3 atom of PLP Schiff base. The average separation between the O3 atom of PLP Schiff base and the phenolic hydrogen atom of Tyr225 is ∼1.85 ˚ A as shown in Table IV. In contrast, in the covalent enolimine tautomer where the O3 atom is covalently linked with the proton, the Tyr225 becomes a hydrogen bond acceptor. The average separation between H atom of PLP Schiff base and phenolic oxygen atom of Tyr225 is ∼3.05 ˚ A. Thus, the present results show that the Tyr225 undergoes a conformational change during the intramolecular proton transfer reaction and forms a stronger hydrogen bond with ketoenamine tautomer as a hydrogen bond donor relative to the enolimine tautomer as a hydrogen bond acceptor. It may be noted that in the ketoenamine form the Tyr225 maintains its proximity with the bridging proton. However, the angle between the phenolic oxygen atom of Tyr225, bridging hydrogen and O3 atom of PLP Schiff base is not ideal for hydrogen bonding.


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The present molecular dynamics simulations reveal that both the tautomers form a strong intramolecular hydrogen bond in the active site of AspAT. The favorable electrostatic as well as hydrogen bonding interactions between the active site residue Asp222 and pyridine leads the latter to be in a protonated state in the active site of AspAT. The active site residues form stronger hydrogen bonds with the ketoenamine tautomer compared to the other. The present results clearly show that the ketoenamine tautomer will be more preferred over the enolimine tautomer in the active site of AspAT.

3.3

Metadynamics simulations The free energy surface for the intramolecular proton transfer reaction of PLP-Asp

Schiff base in the active site of AspAT is shown in Fig. 7 using the metadynamics technique within QM/MM setup. Minimum energy pathway was then traced on this three dimensional surface to estimate the free energy difference between the two configurations. The geometry of the reactant, transition state and product obtained from the metadynamics simulations is shown in Fig. 8. To simulate the transformation from ketoenamine to enolimine tautomer, the coordination number (CN) between an atom with respect to a set of other atoms is used as the collective variable. The CN can be used to detect the presence of a bond between two atoms or for counting the bonds between two different atomic species. In the present calculations, two collective variables were chosen: (a) coordination number of the imino nitrogen (N) atom with respect to the hydrogen atom (H) c[N − H] (b) coordination number of the phenolic oxygen (O3) with respect to hydrogen atom (H) c[O3 − H]. Using these generalized coordinates, the zwitterionic ketoenamine tautomer is characterized by the coordination numbers c[O3 − H] ≈ 1 and c[N − H] ≈ 0, whereas for the covalent enolimine tautomer, the coordination numbers are c[O3 − H] ≈ 0 and c[N − H] ≈ 1. Thus, the trajectories in this two-dimensional reaction subspace spanned by c[O3 − H] and c[N − H] allow to monitor the tautomeric equilibrium in a general way.

The reconstructed free energy surface shows that the zwitterionic ketoenamine Schiff base is more stable than the covalent enolimine Schiff base in the active site of AspAT. The activation barrier for the conversion of zwitterionic ketoenamine tautomer into covalent enolimine tautomer is 7.5 kcal/mol. The free energy difference between the zwitterionic



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tautomer and covalent tautomer is 7 kcal/mol. Note that the values of the coordination number c[N − H] are much larger (mostly between 0.4 and 1.0) during the simulation than their ideal value of 0.0 when hydrogen atom (H) is deprotonated (see the position of the minimum in the energy surface). This means a relatively strong bonding between the imino nitrogen (N) and the proton (H).

The present calculations using the static quantum-chemical approach showed that the zwitterionic ketoenamine tautomer of external PLP-Asp Schiff base corresponds to a local minimum. The classical MD simulations also show that this conformation forms stronger hydrogen bonds with active site residues of AspAT compared to the enolimine configuration. The calculation of proton affinity reveals that the imino nitrogen (N) has a greater tendency to take up a proton compared to the phenolic oxygen. Finally, the result of metadynamics simulations confirm that the ketoenamine tautomer PLP-Asp Schiff base is preferred over the enolimine tautomer in the active site of AspAT. The organization of the active site controls the preference of ketoenamine configuration.

It was postulated that in the active site of PLP dependent enzyme, the protonation of pyridine ring shifts the tautomeric equilibrium from enolimine to ketoenamine state 40 . The present study shows that in the active site of AspAT, the pyridine nitrogen is protonated and the ketoenamine configuration is more preferred. In contrast to the present study, a recent QM/MM study showed that though the pyridine nitrogen is protonated, the enolimine configuration of PLP Schiff base is more stable in the active site of DDC 14 . Another study by the same group showed that in the active site of AlaR, the ketoenamine configuration is preferred although the pyridine nitrogen is unprotonated. Therefore, in the enzyme active site, the protonation state of the pyridine ring is not the only factor which controls the tautomeric equilibrium. There exists some other factors which control the tautomeric equilibrium in the active site of PLP dependent enzymes.

To understand this issue, we compare the hydrogen bonding pattern between the phenolic oxygen of PLP Schiff base and active site residues of AspAT, DDC and AlaR. This is shown in Fig. 9. The figure shows that the phenolic oxygen can form two hydrogen bonds with the active site residues in case of AspAT and AlaR (with Asn194 and Tyr225 in case of AspAT and Arg136 in case of AlaR) while the same can form only one hydrogen 16 Environment ACS Paragon Plus

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bond in case of DDC (with Thr246). The microenvironment created by the AspAT and AlaR favors the ketoenamine configuration while the microenvironment created by the DDC favor the enolimine configuration of the PLP Schiff base. This finding leads us to conclude that the tautomeric equilibrium in the active site of PLP dependent enzymes is strongly dependent on the microenvironment created by the active site residues. If a number of hydrogen bond donating (to the phenolic oxygen) side chains are available in the active site, then the equilibrium will be shifted towards the zwitterionic ketoenamine configuration as in case of AspAT, AlaR else it will prefer the enolimine configuration as in case of DDC. A recent study also showed that the architecture of the active site is perfect complimentary to the substrate and controls the specific substrate binding. It creates a network of interactions which is favorable for the cognate substrate and highly unfavorable for the non-cognate one 75 . The present finding is also supported by the study of the model compound of Schiff base which shows that the tautomeric equilibrium depends on both the protonation state of the pyridine nitrogen as well as intermolecular hydrogen bonds between phenolic oxygen and other proton donating groups.

Overall, the study shows that the protonation state as well as tautomeric equilibrium of PLP-Asp Schiff base are strongly dependent on the microenvironment created by the active site residues of PLP dependent enzymes. If the active site includes such active site residues which can form several intermolecular hydrogen bonds with phenolic oxygen as a hydrogen bond donor, then the equilibrium will be shifted from the covalent enolimine tautomer to the zwitterionic ketoenamine tautomer. This conclusion is also supported by a recent experimental study by Chan-Huot et al. 34 which shows that the formation of OHO intermolecular hydrogen bond to the phenolic oxygen activates the cofactor by shifting the phenolic proton towards the schiff base nitrogen.

4

Summary and conclusions We have carried out quantum chemical calculations and classical as well as hybrid

quantum/classical (QM/MM) molecular dynamics simulations to study the tautomeric equilibrium of PLP-Asp Schiff base in the active site of AspAT. The investigation of the free energy change (∆G) using static quantum mechanical approach in gas phase, in aqueous medium and in active site of AspAT shows that the ketoenamine tautomer is 17 Environment ACS Paragon Plus


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more preferred than the enolimine tautomer. The analysis of the proton affinity reveals that the imino nitrogen (N) has greater tendency to take up a proton compared to the phenolic oxygen (O3). In the active site of AspAT, the pyridine nitrogen remains as protonated and forms strong intermolecular hydrogen bond with Asp222. The classical MD simulations show that the active site of AspAT is more suitable for ketoenamine tautomer compared to the enolimine tautomer. In the zwitterionic form, the anionic phenolic oxygen forms stronger hydrogen bonds with active site residues such as Asn194 and Tyr225 and favor the equilibrium towards ketoenamine configuration. Finally, the metadynamics study confirms these results. The ketoenamine isomer of PLP-Asp Schiff base is preferred with a relative free energy of 7 kcal/mol over the enolimine configuration in the active site of AspAT. The calculated free energy barrier is found to be about 7.5 kcal/mol.

The present study reveals an important factor which controls the tautomeric equilibrium in the active site of PLP dependent enzyme. The presence of hydrogen bond donor groups near the phenolic oxygen of PLP helps to stabilize the zwitterionic isomer of PLP Schiff base and shifts the tautomeric equilibrium towards the ketoenamine isomer. Therefore, intermolecular hydrogen bonding between hydrogen bond donor groups (part of enzyme) and phenolic oxygen (part of PLP) plays a crucial role in the tautomeric equilibrium. If the active site contains more than one hydrogen bond donor groups which can form hydrogen bonds with the phenolic oxygen of PLP (like AspAT or AlaR), then the ketoenamine isomer will be preferred over the other one.


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Table I: The free energy differences between the ketoenamine and enolimine forms for the model tautomerization reaction in the gas phase, aqueous solution and in the active site of AspAT obtained using the B3LYP/6-311+G**//HF/6-311+G** level of calculations. All energies are given in kcal/mol. a Polarizable continuum solvation free energy were calculated at the optimized structures found in the gas phase calculations. b B3LYP/6-311+G**:PM3//HF/6-311+G**:PM3 level of theory is applied to calculate the free energy difference between the ketoenamine and enolimine forms in the active site of AspAT. reaction ∆Ggas ketoenamine → enolimine 8.64

∆GP CM a 8.75

∆Gactivesite b 6.91

Table II: The proton affinity (PA) of phenolic oxygen (O3) of the enolimine tautomer and imino nitrogen (N) of the ketoenamine tautomer of PLP-Asp Schiff base in the gas phase, in aqueous medium and in the active site of AspAT obtained using the B3LYP/6311+G**//HF/6-311+G** level of calculations. a PA in aqueous medium was calculated by employing the PCM model at the optimized structures found in the gas phase calculations. b B3LYP/6-311+G**:PM3//HF/6-311+G**:PM3 level of calculations is applied to calculate the PA in the active site of AspAT. System In Gas Phase In aqueous mediuma In active site of AspATb

Tautomer Ketoenamine enolimine Ketoenamine enolimine Ketoenamine enolimine

PA (kcal/mol) 511.20 502.86 299.78 292.70 326.99 321.11

Table III: Selected average intramolecular structural parameters (separations as well as dihedral angle) for the substrate PLP-Asp Schiff base in ketoenamine tautomer and enolimine tautomer in the active site of AspAT in ˚ A for both A and B chains. The results are obtained from the classical MD simulations. Parameters rO3−N rO3−H rN −H φN −C4A−C4−C3

Ketoenamine (A chain) 2.69 1.98 1.01 -6.92

Ketoenamine (B chain) 2.68 1.96 1.01 -6.85

Enolimine (A chain) 2.64 0.97 1.76 -2.46

Enolimine (B chain) 2.63 0.974 1.73 -1.86

Table IV: Selected intermolecular average separations between the substrate PLPAsp Schiff base (ketoenamine and enolimine tautomers) and active site residues in the active site of AspAT in ˚ A for both A and B chains. The results are obtained from the classical MD simulations.



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PLP-Asp Schiff base O3 O3 O O O3 H O3 H1 N1 H1 N1

Residue H2, Asn194 N, Asn194 H1, Asn194 N, Asn194 H, Tyr225 O, Tyr225 O, Tyr225 Oδ1, Asp222 Oδ1, Asp222 Oδ2, Asp222 Oδ2, Asp222

Ketoenamine (A chain) 1.86 2.78 2.03 2.92 1.84 3.05 2.78 2.22 3.037 1.98 2.92

Ketoenamine (B chain) 1.86 2.76 2.01 2.92 1.86 2.99 2.79 2.28 3.08 1.96 2.91

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Enolimine (A chain) 2.03 2.85 2.02 2.94 3.73 3.07 3.19 2.36 3.18 2.05 2.98

Enolimine (B chain) 2.13 2.88 2.39 3.31 4.42 3.59 3.86 2.18 3.03 2.08 2.98

X-ray

2.63 3.11

3.01 3.38 2.67

Acknowledgment We thank N. Nair and Ravi Tripathi for helpful discussions at the initial stage of this work. Thanks are also due to Arindam Bankura for his help in setting up the QM/MM calculations. We gratefully acknowledge financial support from the Department of Science and Technology (DST), Government of India. S. D. B. is thankful to IIT Kanpur for a postdoctoral fellowship.


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Figure 1: Schematic representation of the PLP-Asp Schiff base with the atom numbering scheme.

Figure 2: Schematic representation of the reactant and product of the tautomeric equilibrium of PLP-Asp Schiff base.


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COO

H2 C

OOC

OOC

H CA

H

H

C4A

C5

C2

H

C5A

C4 C3

H3C

H CA

N

O3

COO

H2C

H

PO3

N

O3

O

C5

C2 H3C

H1

C5A

C4 C3

C6 N1

H

C4A

-2

H

PO3-2 O

C6 N1 H1

(a)

(b)

(c)

(d)

Figure 3: (a) Schematic representation of external PLP-Asp Schiff base in gas phase; (b) Schematic representation of external PLP-Asp Schiff base in aqueous solution; (c) Active site of the crystal structure of the Aspartate aminotransferase complexed with pyridoxyl-aspartic 0 acid 5 monophosphate (PDB code: 1ARG) 48 . The structure is the basis of the model used in the present work. The reactants (PLP-Asp Schiff base) are shown in CPK style. Side chains of the active site residues in close proximity of the reactants as Asn194, Asp222, Tyr225, Lys258, Arg266, Arg386 are shown by bonds. The image is prepared using VMD 76 . (d) Schematic diagram showing hydrogen bonding interactions between the reacting substrates and active side residues. The values show the hydrogen bonding separation found in the X-ray crystal structure. The PLP-Asp Schiff base is colored in blue.



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Figure 4: The root mean square deviation (RMSD) of the protein backbone with respect to the starting structure of the NVT simulations during the empirical force field based MD simulations for (a) N-protonated ketoenamine tautomer (zwitterionic) and (b) O-protonated enolimine tautomer (covalent).


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(a)

(b)

(c) Figure 5: Snapshots of the active site of AspAT with ketoenamine conformation of PLP-Asp Schiff base. The PLP-Asp Schiff base is shown in ball and stick model. Specific active site residues which form hydrogen bonds with the phenolic oxygen (O3) and pyridine nitrogen (N) are shown by thick stick such as (a) Asn194, (b) Asp222 (c) Tyr225. The left panel is for the A-chain and the right panel is for the B chain. The images are prepared using VMD 76 .



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(a)

(b)

(c) Figure 6: Snapshots of the active site of AspAT with enolimine conformation of PLP-Asp Schiff base. The PLP-Asp Schiff base is shown in ball and stick model. Specific active site residues which form hydrogen bonds with the phenolic oxygen (O3) and pyridine nitrogen (N) are shown by thick stick such as (a) Asn194, (b) Asp222 (c) Tyr225. The left panel is for the A-chain and the right panel is for the B chain. The images are prepared using VMD 76 .


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(a)

Enolimine Tautomer 7.5 kcal/mol

Ketoenamine Tautomer (b)

Figure 7: (a) Reconstructed free energy surface for the conversion of N-protonated ketoenamine tautomer (zwitterionic) to O-protonated enolimine tautomer (covalent). The free energies are expressed in kcal/mol. The abscissa represents two CVs; CV1 (c[N − H]: coordination number between nitrogen and hydrogen) and CV2 (c[O3−H]: coordination number between oxygen and hydrogen), see Equation 1 for the definition of coordination number. (b) A schematic diagram of the free energy profile for the same process.



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(a)

(b)

(c) Figure 8: The (a) reactant (b) transition state and (c) product observed during the metadynamics simulation of the tautomeric equilibrium between N-protonated ketoenamine tautomer (zwitterionic) and O-protonated enolimine tautomer (covalent). The images are prepared using 36 Environment VMD 76 . ACS Paragon Plus

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(a)

(b)

(c) Figure 9: Active sites of (a) Ala-racemase (PDB code: 1L6G) 78 (b) AspAT (PDB code: 1ARG) 48 and (c) DDC (PDB code: 1JS3) 79 with the external aldimine (shown in ball and stick representation) and selected active site residues (shown by stick representation). The hydrogen bonding interaction between active site residues and phenolic oxygen (O3) as well as pyridine nitrogen (N) shown by dotted line. The image is prepared using VMD 76 .



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MM Simulation Study of Intramolecular Proton Transfer in

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