Mechanistic Insights into Metal (Pd2+, Co2+, and Zn2+)âÎ²

Article pubs.acs.org/JPCB

Mechanistic Insights into Metal (Pd2+, Co2+, and Zn2+)−β-Cyclodextrin Catalyzed Peptide Hydrolysis: A QM/MM Approach Tingting Zhang,† Xiaoxia Zhu,† and Rajeev Prabhakar* Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, United States S Supporting Information *

ABSTRACT: In this study, mechanistic insights into the hydrolysis of an extremely stable tertiary peptide bond (Ser−Pro) in the Ser-Pro-Phe sequence by an artificial enzyme, metal (Pd2+, Co2+, or Zn2+)−β-cyclodextrin (CD) complex, have been provided. In particular, the exact reaction mechanism, the location of CD (number of −CH2 groups downstream from the metal center), conformation of CD (primary or secondary rim of CD facing the substrate), the number of CD (one or two), and the optimum metal ion (Pd2+, Co2+, or Zn2+) have been suggested using a state-of-the-art hybrid quantum mechanics/ molecular mechanics (QM/MM: B3LYP/Amber) approach. The QM/MM calculations suggest that the internal delivery mechanism is the most energetically feasible for the peptide hydrolysis. The inclusion of a CD ring at two CH2 groups downstream from the metal center can provide 3 × 105 times acceleration in the activity, while the replacement of Pd2+ with Co2+ enhances the rate activity another 3.7 × 104 times.

I. INTRODUCTION The selective hydrolysis of the extremely stable amide or peptide bond ((O)CNH) of peptides and proteins by synthetic metallopeptidases is required in a wide range of biotechnological and industrial applications such as proteomics,1 protein sequencing,1,2 protein engineering,3,4 protein footprinting,5−11 bioethanol production,12 and designing of catalytic drugs.13,14 The half-life for the hydrolysis of peptide bonds is 350−600 years at room temperature and pH 4−8.15 To accomplish this daunting task, nature has designed highly specialized zinc enzymes known as metallopeptidases.16−24 However, despite the availability of a wide range of enzymes and synthetic reagents, currently only a handful of them have been used for the aforementioned applications and with several shortcomings. Therefore, in the past few years, several metal containing (Pd, Pt, Zn, Cu, Co, Fe, Ni, etc.) synthetic analogues of zinc enzymes have been developed to hydrolyze the amide bond under nondenaturing conditions of temperature and pH, but only a few of them are of practical use.13,25−63 The roles of metal ions in peptide hydrolysis have been extensively reviewed.37,64−72 Breslow et al. and others have combined bond cleaving capabilities of metal complexes (M) with the hydrophobic environment of β-cyclodextrin (CD) to design artificial enzymes (M−CD).73−75 In M−CD complexes, the hydrophobic cavity of CD binds the substrate and the metal center cleaves the scissile bond. In comparison to the uncatalyzed reaction, they provided substantial rate accelerations because little entropy and conformational enthalpy were spent in approaching the transition state.76,77 For the first time, the Pd complex (2-Pd), a conjugate of the [Pd(H2O)4]2+ and β© 2014 American Chemical Society

cyclodextrin (CD), 6-S-2-(2-mercaptomethyl)-propane-6-deoxy−β-cyclodextrin diaqua palladium(II), was reported to sequence-specifically cleave the unactivated tertiary Ser−Pro peptide bond in the sequence of Ser-Pro-Phe of the bradykinin substrate at pH 7.0 and 60 °C (Figure 1).78 The measured rate

Figure 1. The experimentally proposed mechanisms for peptide hydrolysis.

constant of 0.07 h−1 for this reaction corresponds to the barrier of 23.8 kcal/mol using the Arrhenius equation (k = Ae−Ea/RT, where A is the pre-exponential factor, Ea is the activation energy, R is the gas constant, and T is the temperature). 2-Pd catalyzes the hydrolysis of the Ser−Pro bond through the following overall reaction: Received: March 4, 2014 Revised: March 28, 2014 Published: March 28, 2014 4106

dx.doi.org/10.1021/jp502229s | J. Phys. Chem. B 2014, 118, 4106−4114

The Journal of Physical Chemistry B

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Figure 2. Structures (in Å) and energies (in kcal/mol) of the optimized reactant, transition state, and product in the QM model for the hydrolysis of the Ser−Pro peptide bond in the Ser-Pro-Phe sequence: (a) external attack mechanism; (b) internal delivery mechanism.

center with the primary rim facing the substrate for n = 2; and (4) the substitution of Pd2+ with biological metal ions (Co2+ and Zn2+). These calculations will afford a deeper understanding concerning the roles of the metal ion and CD cavity on the energetics of this important reaction. They will also provide structures of transition states and short-lived intermediates that cannot be easily observed through existing experimental techniques. In addition, the information gleaned from this study will help to develop the next generation of artificial enzymes for efficient peptide hydrolysis.

Ser(O)CNHPro + H 2O → SerCOOH + NH 2Pro

(1)

The experimentally proposed mechanism of reaction 1 catalyzed by 2-Pd is described in Figure 1. First, the aromatic side chain of the Phe residue of the substrate binds to the cavity of CD through weak hydrophobic interactions and positions the Pd2+ metal center adjacent to the Ser−Pro peptide bond. The Pd2+ ion then coordinates to the carbonyl oxygen atom (OS) of the substrate and activates the amide group toward nucleophilic attack by a water molecule.79−81 After the formation of this complex, the peptide bond cleavage can occur through the following two kinetically indistinguishable mechanisms (Figure 1): (I) Cleavage by external attack: the external solvent water molecule (W1) is activated for hydrolysis. (II) Cleavage by internal delivery: the Pd2+-bound water molecule (W2) is utilized for hydrolysis. However, the exact reaction mechanism and the information regarding the location of CD (number (n) of −CH2 groups downstream from the S atom), the conformation of CD (primary or secondary rim of CD facing the substrate), the number of CD (one or two), and the optimum metal ion (Pd2+, Co2+, or Zn2+) in the 2-Pd complex are currently not available.78 This information is critical to understanding the functioning of this class of artificial enzymes and improving their activities. The available experimental data provides an ideal platform to address these outstanding issues through state-of-the-art theoretical approaches employed in this study. In the first step, the pure QM approach (DFT: B3LYP) is applied to determine the energetically most feasible mechanism for the hydrolysis of the Ser−Pro peptide bond of the Ser-Pro-Phe substrate by the Pd complex without CD (1-Pd) In the next step, the hybrid quantum mechanics/molecular mechanics (QM/MM) method ONIOM: B3LYP/Amber has been applied in a systematic manner to perform the following studies (Figure 1): (1) the primary rim of CD facing the substrate for n = 1 and 2; (2) the secondary rim of CD facing the substrate; (3) inclusion of the second CD molecule to the left of the metal

II. COMPUTATIONAL DETAILS IIa. Method. All QM and QM/MM calculations were carried out using the Gaussian 09 program package.82 The B3LYP/Lanl2dz method was used for the QM only calculations83,84 (with the corresponding Hay−Wadt effective core potential (ECP) for Pd, Co, and Zn85). Frequency calculations at the same level were also performed to characterize the nature of all minima and transition states. The final energies of the optimized structures in the QM calculations were obtained at the B3LYP/{Lanl2dz + d(O, N, S and C) + p(H)} level including thermal corrections. Natural bond orbital (NBO) charges were used to discuss the reactivities of metal complexes. In our two-layer (QM/MM) ONIOM calculations, the system was divided into two parts. A selected model system (including the Pd(II) catalytic center and cleaving peptide bond) where the chemical reactions occur was treated with quantum mechanics (QM, a high-level method), while the remaining part was treated with molecular mechanics (MM, a low-level method). The ONIOM optimization procedure uses macro/microiterations,86 and the electrostatic interactions between the QM and the MM part were treated by mechanical embedding (ME). The present models used charges from ESP (Merz−Kollman) calculations. Charges were calculated for the first stationary point of each model, and the same charges were used in all optimizations with that model. The geometries of reactants, transition states, and products in the QM part were optimized without any symmetry constraints at the B3LYP/ 4107



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Figure 3. Structures (in Å) and energies (in kcal/mol) of the optimized reactant, transition state, and product for 2-Pd.

Lanl2dz level of theory83,84 (with the corresponding Hay− Wadt effective core potential (ECP) for Pd, Co, and Zn85), and the MM part was calculated using the Amber force field. The final energies of the optimized structures were further improved by performing single point calculations including additional d and p polarization functions for O (α = 0.96), N (α = 0.74), C (α = 0.59), S (α = 0.50), and H (α = 0.36) atoms, respectively (taken from the EMSL’s Gaussian basis set library), in the basis set used for optimizations. Frequency calculations were performed at the same (B3LYP/Lanl2dz:Amber) level as used for the geometry optimizations to obtain zero-point vibrational (unscaled), thermal (at 298.15 K and 1 atm), and entropy corrections (at 298.15 K). Transition states were confirmed to have only one imaginary frequency corresponding to the reaction coordinate. To test the accuracy of the B3LYP method, the barrier of the reaction computed using this functional was compared with the one calculated using the MPW9187 and M06-L functional88 for the 2-Pd complex. It was found that, in comparison to the B3LYP functional (barrier = 32.8 kcal/mol), the MPW91 and M06-L functionals increased the barrier by 0.1 and 3.4 kcal/ mol, respectively. IIb. Models. The available experimental information was fully incorporated in building models for the calculations. In the QM only study, the Pd complex (without β-CD) and substrate, a tripeptide (Ser-Pro-Phe) model (1-PdE and 1-Pd), were utilized. While in the ONIOM study, Pd complexed with β-CD (2-PdE and 2-Pd) and the tripeptide as the substrate were considered. For the β-CD dimer case (2-Pd2CD), a tetrapeptide sequence (Phe-Ser-Pro-Phe) was used so as to provide double binding sites. The initial coordinates of β-CD were taken from the three-dimensional crystal structure.89 The charge on all the systems is +2. The Pd- and Zn-containing complexes exist in the singlet spin state, while the Co(II) complex in the high spin quartet spin state.

III. RESULTS AND DISCUSSION IIIa. Mechanism of Peptide Hydrolysis by the Pd Complex without the CD Ring (1-Pd). The peptide bonds can be cleaved through the following two mechanisms: (1) external attack mechanism and (2) internal delivery mechanism. In the external attack mechanism (Figure 2a), the Pd(II) complex binds to the substrate through the coordination between the Pd(II) ion and the carbonyl oxygen atom OS in 1PdRE. The interaction between the Pd(II) ion and OS atom increases the electrophilicity of the CS and activates it for an attack by an external water molecule (W1).90 In the first step, in a concerted fashion, the NS atom of the peptide bond abstracts a proton from the external water molecule (W1), and the hydroxyl group (−O2H) makes a nucleophilic attack on the CS of the peptide bond. This process leads to the cleavage of the CS−NS peptide bond with the computed barrier 51.9 kcal/mol. The optimized transition state for this process (1-PdTSE) is shown in Figure 2a. All the relevant bond distances indicate that this process is concerted (O2−CS = 1.69 Å, H2−NS = 1.33 Å, O2−H2 = 1.21 Å, NS−CS = 1.58 Å). The DFT calculations suggest that, in the external attack mechanism, the calculated barriers are prohibitively high (>50.0 kcal/mol) for the cleavage of the peptide bonds, and therefore, this mechanism is ruled out. In the first step of the internal delivery mechanism, Pd(II) also functions as a Lewis acid and binds to the carbonyl oxygen atom (OS) with a distance of 2.08 Å (Figure 2b, 1-PdR). Instead of using an external water molecule, a water molecule (W2) bound to the Pd(II) ion is activated. In the transition state (1PdTS), the O1−H1 bond is broken, and the proton H1 is transferred to the NS of the peptide bond. This process occurs concomitantly with the transfer of the hydroxyl group (−O1H) to the CS atom and leads to the cleavage of the peptide bond with an energy barrier of 40.2 kcal/mol. This barrier is 11.7 kcal/mol lower than the barrier in the external attack mechanism (51.9 kcal/mol). This reduction of the barrier is 4108



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Figure 4. Structures (in Å) and energies (in kcal/mol) of the optimized reactant, transition state, and product for 2′-Pd.

caused by the substantial lowering of the pKa value of the W2 water molecule due to the binding to the Pd2+ ion. Since the internal delivery mechanism is found to be significantly more energetically feasible than the external attack mechanism, only this mechanism (in the concerted form) is further utilized to investigate the effect of CD on the activity of the 2-Pd complex through QM/MM calculations. IIIb. Mechanism of Peptide Hydrolysis with the Inclusion of the CD Ring. The Pd catalyst and substrate (Ser-Pro-Phe) first form a host−guest complex through hydrophobic interactions between CD and Phe residue and the coordination of the Pd2+ ion with the carbonyl oxygen (OS) of the scissile peptide bond (CS−NS), Figures 3 and 4. From the reactant, the metal-bound water molecule (H1O1H) is activated and in a concerted manner the H1 proton and −O1H hydroxyl ion are transferred to the NS and CS atoms of the scissile peptide bond, respectively. This process leads to the cleavage of the peptide bond, and separated metal-bound carboxyl and amine terminals are created (2-PdP). Since the Pd metal center is attached to the C6 atom of the glucose unit of CD, there are two possible locations for the CD binding (n = 1 or 2). On the basis of the position of CD, the complexes at n = 1 and 2 are referred to as 2′-Pd and 2-Pd, respectively. The computed barrier (45.8 kcal/mol) for 2′-Pd is 13.0 kcal/mol higher than the one for 2-Pd (32.8 kcal/mol), Table 1. In the presence of CD at n = 2 also, the barrier (39.0 kcal/mol) for the external delivery mechanism (2-PdE, Figure 5) is 6.2 kcal/ mol higher than the barrier (32.8 kcal/mol) for the internal delivery mechanism (2-Pd, Figure 3). However, the exact protonation state of the metal-bound water molecule (H2O or −OH) in the internal delivery mechanism is not known. A metal-bound hydroxyl group complex (2-PdOH) will utilize a stepwise mechanism for peptide hydrolysis (Figure S1, Supporting Information). According to this mechanism, a nucleophilic attack of the −O1H1 group on the CS atom of the substrate leads to the creation of a tetrahederal intermediate (2PdINOH) in which both O1 and OS atoms are coordinated to

Table 1. Energetics of Peptide Hydrolysis complex

barrier (kcal/mol)

exothermicity (kcal/mol)

1-PdE 1-Pd 2-PdE 2-Pd 2-PdOH 2′-Pd 2′-PdS 2-Pd2CD 2-Co 2-Zn

51.9 40.2 39.0 32.8 33.5 45.8 37.0 35.6 26.6 35.9

−3.3 1.4 6.2 −7.7 −6.7 8.9 9.2 −14.8 −26.8 5.8

the Pd ion (Figure S1, Supporting Information). This process occurs with a barrier of 19.8 kcal/mol and is endothermic by 18.6 kcal/mol. In the next step, a transfer of the H1 proton to the NS atom of the CS−NS peptide bond cleaves the bond. From the reactant, this concerted and rate-limiting process takes place with a barrier of 33.5 kcal/mol. The barrier using this mechanism is only slightly higher by 0.7 kcal/mol than the barrier of 32.8 kcal/mol for the concerted mechanism utilized by 2-Pd. On the other hand, the barrier of 32.8 kcal/mol for 2-Pd is 7.4 kcal/mol lower than the one without CD (1-Pd), Table 1. However, this barrier is 9.0 kcal/mol higher than the measured value.78 This overestimation could be partially attributed to the measurement of the rate constants at a much higher temperature (60 °C), whereas our calculations were performed at room temperature (25 °C). Due to the temperature dependence of the pre-exponential constant in the Arrhenius equation, it is not possible to accurately estimate the measured barrier at 25 °C. The binding of CD induces substantial structural changes and increases the Pd−OS distance by 0.03 Å (Pd−OS = 2.11 Å) and brings the water nucleophile closer to the scissile peptide bond (CS−NS). In 2-PdR (Figure 3), the electronic charges on the Pd ion and OS atoms are increased by 0.03 and 0.02 e, respectively (Pd = 0.45 e and OS = −0.72 e). 4109



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Figure 5. Structures (in Å) and energies (in kcal/mol) of the optimized reactant, transition state, and product for 2-PdE.

Figure 6. Structures (in Å) and energies (in kcal/mol) of the optimized reactant, transition state, and product for 2′-PdS.

shortens the bond distance between the Pd2+ ion and water nucleophile (Pd−O1) and pushes the nucleophile and peptide bond (CS−NS) away from each other (Pd−O1 = 2.08 Å, O1− CS = 3.37 Å, and H1−NS = 3.12 Å), Figure 4. In comparison to 2-Pd, the Pd−O1 distance shrinks by 0.03 Å and the O1−CS and H1−NS distances elongate by 0.17 and 0.02 Å, respectively (Figure 3). All of these changes lower the peptidase activity of the complex. These results suggest that the inclusion of the CD ring at the n = 2 locations can provide 3 × 105 times acceleration in the hydrolytic activity. Therefore, only this binding location (n = 2) is used in the remaining calculations. IIIc. Secondary Rim of CD Facing the Substrate (2′PdS). The binding of the secondary rim of CD with the Pd2+

These changes enhance the Lewis acidity of the metal center and increase its peptidase activity. In the optimized transition state (2-PdTS), all the corresponding distances indicate that this process is concerted (O1−CS = 2.37 Å, O1−H1 = 2.01 Å, H1− NS = 1.05 Å, and CS−NS = 1.54 Å). The aforementioned structural changes induced by the binding of CD create much better conditions for the cleavage of the peptide bond. The effect of CD on the energetics of the reaction is very surprising, because it was previously believed that CD only provides specificity to the Pd complex.78 On the other hand, for the n = 1 system (2′-Pd, Figure 4), the computed barrier of 45.8 kcal/ mol is 5.6 kcal/mol higher than the barrier (40.2 kcal/mol) in the absence of CD (1-Pd, Figure 2), Table 1. The reason for this increase could be that the presence of CD in 2′-Pd 4110



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Figure 7. Structures (in Å) and energies (in kcal/mol) of the optimized reactant, transition state, and product for 2-Pd2CD.

Figure 8. Structures (in Å) and energies (in kcal/mol) of the optimized reactant, transition state, and product for 2-Co.

structural differences raise the barrier of the reaction by 4.2 kcal/mol as compared with the one for the 2-Pd system (Table 1). IIId. Inclusion of the Second CD Ring at n = 2 (2Pd2CD). The effect of the second CD on the energetics of the reaction was investigated by including an additional CD ring to the left of the metal center and a Phe residue in the substrate (Phe-Ser-Pro-Phe sequence) in 2-Pd. In the reactant (2PdR2CD, Figure 7) of this complex, both Phe residues interact with the CD rings through hydrophobic interactions. In 2-

center also forms the host−guest complex with the substrate (2′-PdS, Figure 6). This complex can exist only for n = 1, because of the binding of the Pd complex to the CD rim through the C2 atom of the glucose unit on the CH−OH group. However, due to the larger cavity of the secondary rim, the interactions of the Phe residue of the substrate with CD are much weaker than those in 2-Pd. Furthermore, in the reactant (2′-PdRS, Figure 6), the Pd−OS and O1−CS bond distances are decreased and increased by 0.03 and 0.08 Å, respectively (Pd− OS = 2.08 Å and O1−CS = 3.28 Å), compared to 2-PdR. These 4111



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PdR2CD, the Pd−O1 and O1−CS distances are 0.04 and 0.16 Å shorter, respectively, while the Pd−OS bond is 0.02 Å longer than the corresponding distances in the reactant (2-PdR) with one CD ring. Rather interestingly, in comparison to 2-Pd, the inclusion of the second CD does not provide further rate acceleration and actually increases the barrier slightly by 2.8 kcal/mol, i.e., 35.6 kcal/mol from 2-PdR2CD (Table 1). It is noteworthy that, under experimental conditions, the presence of two CD rings should facilitate a rapid formation of the substrate−catalyst complex and increase the rate of reaction. However, in the reactant (2-PdR2CD) used in this study, the substrate is already bound to the metal complex. IIIe. Substitution of Pd with Co and Zn in 2-Pd. To study the role of the metal ion in the reaction, the nonbiological Pd2+ ion in 2-Pd was substituted with biological Co2+ and Zn2+ metals (2-Co and 2-Zn, respectively). For 2-Co, the high spin quarter state was found to be 4.2 kcal/mol (in the gas phase) lower in energy than the doublet state. In the reactant of the Co2+ variant (2-CoR, Figure 8), all the key distances, Co−O1, O1−CS, and Co−OS bond distances, are substantially shorter by 0.07, 0.06, and 0.13 Å, respectively, than the corresponding distances in the Pd2+ containing counterpart (2-PdR), Table 2.

replacement of Pd2+ that can further provide 3.7 × 104 times rate enhancement. The results reported in this study provide a deeper understanding of the functioning of these artificial enzymes and suggest modifications to improve their activities.

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* Supporting Information (a) Complete ref 82; (b) Figures S1 and S2; (c) Tables S1− S32: Cartesian coordinates (in Å) of all the optimized structures. This material is available free of charge via the Internet at http://pubs.acs.org.

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2-PdRE

metal−S1

*E-mail: [email protected]. Phone: 305-284-9372. Fax: 305-2844571. Author Contributions †

T.Z. and X.Z. contributed equally to the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the grant of the National Science Foundation (Grant Number 1152846) to R.P. We thank Thomas Paul and Ali Sawani for their assistance in the preparation of the manuscript.

metal−S2

2-PdR 2′-PdR 2′-PdRS 2-PdR2CD 2-CoR 2-ZnR transition state

2.41 2.40 2.41 2.41 2.42 2.47 2.49 metal−S1

2.41 2.42 2.42 2.41 2.42 2.48 2.61 metal−S2

2-PdTSE 2-PdTS 2′-PdTS 2′-PdTSS 2-PdTS2CD 2-CoTS 2-ZnTS

2.40 2.44 2.42 2.41 2.41 2.42 2.48

2.41 2.40 2.41 2.41 2.42 2.49 2.54

AUTHOR INFORMATION

Corresponding Author

Table 2. Metal−Ligand Distances (in Å) for All Metal−CD Complexes reactant

ASSOCIATED CONTENT

S

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ABBREVIATIONS CD, cyclodextrin; DFT, density functional theory; QM/MM, quantum mechanics/molecular mechanics; 2-Pd, primary rim of CD facing the substrate for n = 2; 2′-PdS, secondary rim of CD facing the substrate for n = 1; 2-Pd2CD, inclusion of the second CD ring at n = 2

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REFERENCES

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In the transition state (2-CoTS, Figure 8) of the Co complex, the scissile peptide bond (CS−NS) is also 0.01 Å more activated than that in 2-PdTS. In addition, there is a better driving force for the reaction in the Co complex and the reaction from 2-CoR is 19.1 kcal/mol more exothermic than the one from 2-PdR. Due to these differences, in comparison to the 2-Pd complex (32.8 kcal/mol), the barrier for the reaction is lower by 6.2 kcal/mol, i.e., 26.6 kcal/mol for 2-Co (Table 1). On the other hand, for 2-Zn, the barrier is increased by 3.1 kcal/mol (35.9 kcal/mol) as compared with that of 2-Pd (Table 1).

IV. CONCLUSIONS The major conclusions of this study are as follows: (1) the internal delivery mechanism is the most energetically feasible for the peptide hydrolysis and both concerted and stepwise pathways proceed through similar barriers; (2) the optimum position for the inclusion of the CD ring is n = 2 and it can provide 3 × 105 times acceleration in the activity of 2-Pd; (3) the substrate should interact through the primary rim of CD; and (4) among Pd2+, Co2+, and Zn2+, Co2+ is the most suitable 4112



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