Mechanistic Insights into Metal (Pd2+, Co2+, and Zn2+)−β-CyclodextrinCatalyzed Peptide Hydrolysis: A QM/MM ApproachTingting 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 anextremely stable tertiary peptide bond (Ser−Pro) in the Ser-Pro-Phe sequenceby an artificial enzyme, metal (Pd2+, Co2+, or Zn2+)−β-cyclodextrin (CD)complex, have been provided. In particular, the exact reaction mechanism, thelocation 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+, orZn2+) have been suggested using a state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM: B3LYP/Amber) approach. The QM/MMcalculations suggest that the internal delivery mechanism is the mostenergetically feasible for the peptide hydrolysis. The inclusion of a CD ringat 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 orpeptide bond ((O)CNH) of peptides and proteinsby synthetic metallopeptidases is required in a wide range ofbiotechnological and industrial applications such as proteo-mics,1 protein sequencing,1,2 protein engineering,3,4 proteinfootprinting,5−11 bioethanol production,12 and designing ofcatalytic drugs.13,14 The half-life for the hydrolysis of peptidebonds is 350−600 years at room temperature and pH 4−8.15To accomplish this daunting task, nature has designed highlyspecialized zinc enzymes known as metallopeptidases.16−24

However, despite the availability of a wide range of enzymesand synthetic reagents, currently only a handful of them havebeen used for the aforementioned applications and with severalshortcomings. Therefore, in the past few years, several metalcontaining (Pd, Pt, Zn, Cu, Co, Fe, Ni, etc.) syntheticanalogues of zinc enzymes have been developed to hydrolyzethe amide bond under nondenaturing conditions of temper-ature and pH, but only a few of them are of practicaluse.13,25−63 The roles of metal ions in peptide hydrolysis havebeen extensively reviewed.37,64−72

Breslow et al. and others have combined bond cleavingcapabilities of metal complexes (M) with the hydrophobicenvironment of β-cyclodextrin (CD) to design artificialenzymes (M−CD).73−75 In M−CD complexes, the hydro-phobic cavity of CD binds the substrate and the metal centercleaves the scissile bond. In comparison to the uncatalyzedreaction, they provided substantial rate accelerations becauselittle entropy and conformational enthalpy were spent inapproaching the transition state.76,77 For the first time, the Pdcomplex (2-Pd), a conjugate of the [Pd(H2O)4]

2+ and β-

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

constant of 0.07 h−1 for this reaction corresponds to the barrierof 23.8 kcal/mol using the Arrhenius equation (k = Ae−Ea/RT,where A is the pre-exponential factor, Ea is the activationenergy, 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, 2014Revised: March 28, 2014Published: March 28, 2014

Figure 1. The experimentally proposed mechanisms for peptidehydrolysis.

Article

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+

→ +

Ser (O )C NH Pro H O

Ser COOH NH Pro2

2 (1)

The experimentally proposed mechanism of reaction 1catalyzed by 2-Pd is described in Figure 1. First, the aromaticside chain of the Phe residue of the substrate binds to the cavityof CD through weak hydrophobic interactions and positionsthe 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 towardnucleophilic attack by a water molecule.79−81 After theformation of this complex, the peptide bond cleavage canoccur through the following two kinetically indistinguishablemechanisms (Figure 1): (I) Cleavage by external attack: theexternal solvent water molecule (W1) is activated forhydrolysis. (II) Cleavage by internal delivery: the Pd2+-boundwater molecule (W2) is utilized for hydrolysis. However, theexact reaction mechanism and the information regarding thelocation of CD (number (n) of −CH2 groups downstreamfrom the S atom), the conformation of CD (primary orsecondary 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 Thisinformation is critical to understanding the functioning of thisclass of artificial enzymes and improving their activities.The available experimental data provides an ideal platform to

address these outstanding issues through state-of-the-arttheoretical approaches employed in this study. In the firststep, the pure QM approach (DFT: B3LYP) is applied todetermine the energetically most feasible mechanism for thehydrolysis of the Ser−Pro peptide bond of the Ser-Pro-Phesubstrate by the Pd complex without CD (1-Pd) In the nextstep, the hybrid quantum mechanics/molecular mechanics(QM/MM) method ONIOM: B3LYP/Amber has been appliedin a systematic manner to perform the following studies (Figure1): (1) the primary rim of CD facing the substrate for n = 1 and2; (2) the secondary rim of CD facing the substrate; (3)inclusion of the second CD molecule to the left of the metal

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 under-standing concerning the roles of the metal ion and CD cavityon the energetics of this important reaction. They will alsoprovide structures of transition states and short-livedintermediates that cannot be easily observed through existingexperimental techniques. In addition, the information gleanedfrom this study will help to develop the next generation ofartificial enzymes for efficient peptide hydrolysis.

II. COMPUTATIONAL DETAILSIIa. Method. All QM and QM/MM calculations were

carried out using the Gaussian 09 program package.82 TheB3LYP/Lanl2dz method was used for the QM onlycalculations83,84 (with the corresponding Hay−Wadt effectivecore potential (ECP) for Pd, Co, and Zn85). Frequencycalculations at the same level were also performed tocharacterize the nature of all minima and transition states.The final energies of the optimized structures in the QMcalculations were obtained at the B3LYP/{Lanl2dz + d(O, N, Sand C) + p(H)} level including thermal corrections. Naturalbond orbital (NBO) charges were used to discuss thereactivities 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 peptidebond) where the chemical reactions occur was treated withquantum mechanics (QM, a high-level method), while theremaining part was treated with molecular mechanics (MM, alow-level method). The ONIOM optimization procedure usesmacro/microiterations,86 and the electrostatic interactionsbetween the QM and the MM part were treated by mechanicalembedding (ME). The present models used charges from ESP(Merz−Kollman) calculations. Charges were calculated for thefirst stationary point of each model, and the same charges wereused in all optimizations with that model. The geometries ofreactants, transition states, and products in the QM part wereoptimized without any symmetry constraints at the B3LYP/

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 ofthe Ser−Pro peptide bond in the Ser-Pro-Phe sequence: (a) external attack mechanism; (b) internal delivery mechanism.

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Lanl2dz level of theory83,84 (with the corresponding Hay−Wadt effective core potential (ECP) for Pd, Co, and Zn85), andthe MM part was calculated using the Amber force field. Thefinal energies of the optimized structures were further improvedby performing single point calculations including additional dand 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 basisset used for optimizations. Frequency calculations wereperformed at the same (B3LYP/Lanl2dz:Amber) level asused for the geometry optimizations to obtain zero-pointvibrational (unscaled), thermal (at 298.15 K and 1 atm), andentropy corrections (at 298.15 K). Transition states wereconfirmed to have only one imaginary frequency correspondingto the reaction coordinate.To test the accuracy of the B3LYP method, the barrier of the

reaction computed using this functional was compared with theone calculated using the MPW9187 and M06-L functional88 forthe 2-Pd complex. It was found that, in comparison to theB3LYP functional (barrier = 32.8 kcal/mol), the MPW91 andM06-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 theQM only study, the Pd complex (without β-CD) and substrate,a tripeptide (Ser-Pro-Phe) model (1-PdE and 1-Pd), wereutilized. While in the ONIOM study, Pd complexed with β-CD(2-PdE and 2-Pd) and the tripeptide as the substrate wereconsidered. For the β-CD dimer case (2-Pd2CD), a tetrapeptidesequence (Phe-Ser-Pro-Phe) was used so as to provide doublebinding sites. The initial coordinates of β-CD were taken fromthe three-dimensional crystal structure.89 The charge on all thesystems is +2. The Pd- and Zn-containing complexes exist inthe singlet spin state, while the Co(II) complex in the high spinquartet spin state.

III. RESULTS AND DISCUSSION

IIIa. Mechanism of Peptide Hydrolysis by the PdComplex without the CD Ring (1-Pd). The peptide bondscan 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 coordinationbetween the Pd(II) ion and the carbonyl oxygen atom OS in 1-PdR

E. The interaction between the Pd(II) ion and OS atomincreases the electrophilicity of the CS and activates it for anattack by an external water molecule (W1).90 In the first step, ina concerted fashion, the NS atom of the peptide bond abstractsa proton from the external water molecule (W1), and thehydroxyl group (−O2H) makes a nucleophilic attack on the CS

of the peptide bond. This process leads to the cleavage of theCS−NS peptide bond with the computed barrier 51.9 kcal/mol.The optimized transition state for this process (1-PdTS

E) isshown in Figure 2a. All the relevant bond distances indicatethat this process is concerted (O2−CS = 1.69 Å, H2−NS = 1.33Å, O2−H2 = 1.21 Å, NS−CS = 1.58 Å). The DFT calculationssuggest that, in the external attack mechanism, the calculatedbarriers are prohibitively high (>50.0 kcal/mol) for the cleavageof the peptide bonds, and therefore, this mechanism is ruledout.In the first step of the internal delivery mechanism, Pd(II)

also functions as a Lewis acid and binds to the carbonyl oxygenatom (OS) with a distance of 2.08 Å (Figure 2b, 1-PdR). Insteadof using an external water molecule, a water molecule (W2)bound to the Pd(II) ion is activated. In the transition state (1-PdTS), the O1−H1 bond is broken, and the proton H1 istransferred to the NS of the peptide bond. This process occursconcomitantly with the transfer of the hydroxyl group (−O1H)to the CS atom and leads to the cleavage of the peptide bondwith an energy barrier of 40.2 kcal/mol. This barrier is 11.7kcal/mol lower than the barrier in the external attackmechanism (51.9 kcal/mol). This reduction of the barrier is

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

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caused by the substantial lowering of the pKa value of the W2water 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 attackmechanism, only this mechanism (in the concerted form) isfurther utilized to investigate the effect of CD on the activity ofthe 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 throughhydrophobic interactions between CD and Phe residue andthe coordination of the Pd2+ ion with the carbonyl oxygen (OS)of the scissile peptide bond (CS−NS), Figures 3 and 4. Fromthe reactant, the metal-bound water molecule (H1O1H) isactivated and in a concerted manner the H1 proton and −O1Hhydroxyl ion are transferred to the NS and CS atoms of thescissile peptide bond, respectively. This process leads to thecleavage of the peptide bond, and separated metal-boundcarboxyl and amine terminals are created (2-PdP). Since the Pdmetal center is attached to the C6 atom of the glucose unit ofCD, there are two possible locations for the CD binding (n = 1or 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. Thecomputed barrier (45.8 kcal/mol) for 2′-Pd is 13.0 kcal/molhigher than the one for 2-Pd (32.8 kcal/mol), Table 1. In thepresence of CD at n = 2 also, the barrier (39.0 kcal/mol) forthe external delivery mechanism (2-PdE, Figure 5) is 6.2 kcal/mol higher than the barrier (32.8 kcal/mol) for the internaldelivery mechanism (2-Pd, Figure 3). However, the exactprotonation state of the metal-bound water molecule (H2O or−OH) in the internal delivery mechanism is not known. Ametal-bound hydroxyl group complex (2-PdOH) will utilize astepwise mechanism for peptide hydrolysis (Figure S1,Supporting Information). According to this mechanism, anucleophilic attack of the −O1H1 group on the CS atom of thesubstrate leads to the creation of a tetrahederal intermediate(2PdIN

OH) in which both O1 and OS atoms are coordinated to

the Pd ion (Figure S1, Supporting Information). This processoccurs with a barrier of 19.8 kcal/mol and is endothermic by18.6 kcal/mol. In the next step, a transfer of the H1 proton tothe NS atom of the CS−NS peptide bond cleaves the bond.From the reactant, this concerted and rate-limiting processtakes place with a barrier of 33.5 kcal/mol. The barrier usingthis mechanism is only slightly higher by 0.7 kcal/mol than thebarrier of 32.8 kcal/mol for the concerted mechanism utilizedby 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 measuredvalue.78 This overestimation could be partially attributed to themeasurement of the rate constants at a much highertemperature (60 °C), whereas our calculations were performedat room temperature (25 °C). Due to the temperaturedependence of the pre-exponential constant in the Arrheniusequation, it is not possible to accurately estimate the measuredbarrier at 25 °C. The binding of CD induces substantialstructural changes and increases the Pd−OS distance by 0.03 Å(Pd−OS = 2.11 Å) and brings the water nucleophile closer tothe scissile peptide bond (CS−NS). In 2-PdR (Figure 3), theelectronic charges on the Pd ion and OS atoms are increased by0.03 and 0.02 e, respectively (Pd = 0.45 e and OS = −0.72 e).

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

Table 1. Energetics of Peptide Hydrolysis

complex barrier (kcal/mol) exothermicity (kcal/mol)

1-PdE 51.9 −3.31-Pd 40.2 1.42-PdE 39.0 6.22-Pd 32.8 −7.72-PdOH 33.5 −6.72′-Pd 45.8 8.92′-PdS 37.0 9.22-Pd2CD 35.6 −14.82-Co 26.6 −26.82-Zn 35.9 5.8

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These changes enhance the Lewis acidity of the metal centerand increase its peptidase activity. In the optimized transitionstate (2-PdTS), all the corresponding distances indicate that thisprocess is concerted (O1−CS = 2.37 Å, O1−H1 = 2.01 Å, H1−NS = 1.05 Å, and CS−NS = 1.54 Å). The aforementionedstructural changes induced by the binding of CD create muchbetter conditions for the cleavage of the peptide bond. Theeffect of CD on the energetics of the reaction is very surprising,because it was previously believed that CD only providesspecificity 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) inthe absence of CD (1-Pd, Figure 2), Table 1. The reason forthis increase could be that the presence of CD in 2′-Pd

shortens the bond distance between the Pd2+ ion and waternucleophile (Pd−O1) and pushes the nucleophile and peptidebond (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 to2-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 ofthe complex.These results suggest that the inclusion of the CD ring at the

n = 2 locations can provide 3 × 105 times acceleration in thehydrolytic 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+

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.

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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 rimthrough the C2 atom of the glucose unit on the CH−OHgroup. However, due to the larger cavity of the secondary rim,the interactions of the Phe residue of the substrate with CD aremuch weaker than those in 2-Pd. Furthermore, in the reactant(2′-PdRS, Figure 6), the Pd−OS and O1−CS bond distances aredecreased 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

structural differences raise the barrier of the reaction by 4.2kcal/mol as compared with the one for the 2-Pd system (Table1).

IIId. Inclusion of the Second CD Ring at n = 2 (2-Pd2CD). The effect of the second CD on the energetics of thereaction was investigated by including an additional CD ring tothe left of the metal center and a Phe residue in the substrate(Phe-Ser-Pro-Phe sequence) in 2-Pd. In the reactant (2-PdR

2CD, Figure 7) of this complex, both Phe residues interactwith the CD rings through hydrophobic interactions. In 2-

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.

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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 longerthan the corresponding distances in the reactant (2-PdR) withone CD ring. Rather interestingly, in comparison to 2-Pd, theinclusion of the second CD does not provide further rateacceleration and actually increases the barrier slightly by 2.8kcal/mol, i.e., 35.6 kcal/mol from 2-PdR

2CD (Table 1). It isnoteworthy that, under experimental conditions, the presenceof two CD rings should facilitate a rapid formation of thesubstrate−catalyst complex and increase the rate of reaction.However, in the reactant (2-PdR

2CD) used in this study, thesubstrate 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 nonbiologicalPd2+ ion in 2-Pd was substituted with biological Co2+ and Zn2+

metals (2-Co and 2-Zn, respectively). For 2-Co, the high spinquarter state was found to be 4.2 kcal/mol (in the gas phase)lower in energy than the doublet state. In the reactant of theCo2+ variant (2-CoR, Figure 8), all the key distances, Co−O1,O1−CS, and Co−OS bond distances, are substantially shorter by0.07, 0.06, and 0.13 Å, respectively, than the correspondingdistances in the Pd2+ containing counterpart (2-PdR), Table 2.

In the transition state (2-CoTS, Figure 8) of the Co complex,the scissile peptide bond (CS−NS) is also 0.01 Å more activatedthan that in 2-PdTS. In addition, there is a better driving forcefor the reaction in the Co complex and the reaction from 2-CoRis 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 6.2kcal/mol, i.e., 26.6 kcal/mol for 2-Co (Table 1). On the otherhand, for 2-Zn, the barrier is increased by 3.1 kcal/mol (35.9kcal/mol) as compared with that of 2-Pd (Table 1).

IV. CONCLUSIONSThe major conclusions of this study are as follows: (1) theinternal delivery mechanism is the most energetically feasible forthe peptide hydrolysis and both concerted and stepwisepathways proceed through similar barriers; (2) the optimumposition for the inclusion of the CD ring is n = 2 and it canprovide 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

replacement of Pd2+ that can further provide 3.7 × 104 timesrate enhancement. The results reported in this study provide adeeper understanding of the functioning of these artificialenzymes and suggest modifications to improve their activities.

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

■ AUTHOR INFORMATIONCorresponding Author*E-mail: rpr@miami.edu. Phone: 305-284-9372. Fax: 305-284-4571.Author Contributions†T.Z. and X.Z. contributed equally to the manuscript.NotesThe authors declare no competing financial interest.

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

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

■ REFERENCES(1) Thomas, J. J.; Bakhtiar, R.; Siuzdak, G. Mass Spectrometry inViral Proteomics. Acc. Chem. Res. 2000, 33, 179−187.(2) Lewis, J. K.; Bendahmane, M.; Smith, T. J.; Beachy, R. N.;Siuzdak, G. Identification of Viral Mutants by Mass Spectrometry.Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8596−8601.(3) Thorner, J.; Emr, S. D.; Abelson, J. N. Applications of ChimericGenes and Hybrid Proteins, Part A: Gene Expression and ProteinPurification. Methods Enzymol. 2000, 2000, 326.(4) Wallace, C. J. A. Protein Engineering by Semisynthesis; CRC Press:Boca Raton, FL, 2000.(5) Greiner, D. P.; Hughes, K. A.; Gunasekera, A. H.; Meares, C. F.Binding of the Sigma 70 Protein to the Core Subunits of EscherichiaColi RNA Polymerase, Studied by Iron-EDTA Protein Footprinting.Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 71−75.(6) Heyduk, T.; Heyduk, E.; Severinov, K.; Tang, H.; Ebright, R. H.Determinants of RNA Polymerase Alpha Subunit for Interaction withBeta, Beta’, and Sigma Subunits: Hydroxyl-Radical Protein Foot-printing. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10162−10166.(7) Colland, F.; Fujita, N.; Ishihama, A.; Kolb, A. The Interactionbetween SigmaS, the Stationary Phase Sigma Factor, and the CoreEnzyme of Escherichia Coli RNA Polymerase. Genes Cells 2002, 7,233−247.(8) Cheng, X.; Shaltiel, S.; Taylor, S. S. Mapping Substrate-InducedConformational Changes in cAMP-Dependent Protein Kinase byProtein Footprinting. Biochemistry 1998, 37, 14005−14013.(9) Baichoo, N.; Heyduk, T. Mapping Cyclic Nucleotide-InducedConformational Changes in CyclicAMP Receptor Protein by a ProteinFootprinting Technique Using Different Chemical Proteases. ProteinSci. 1999, 8, 518−528.

Table 2. Metal−Ligand Distances (in Å) for All Metal−CDComplexes

reactant metal−S1 metal−S2

2-PdRE 2.41 2.41

2-PdR 2.40 2.422′-PdR 2.41 2.422′-PdRS 2.41 2.412-PdR

2CD 2.42 2.422-CoR 2.47 2.482-ZnR 2.49 2.61

transition state metal−S1 metal−S2

2-PdTSE 2.40 2.41

2-PdTS 2.44 2.402′-PdTS 2.42 2.412′-PdTSS 2.41 2.412-PdTS

2CD 2.41 2.422-CoTS 2.42 2.492-ZnTS 2.48 2.54

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