Catalytic Mechanism of Class B2 Metallo-β-lactamase
2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês
10.1074/jbc.m512517200
ISSN1083-351X
AutoresDingguo Xu, Daiqian Xie, Hua Guo,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoThe initial nucleophilic substitution step of biapenem hydrolysis catalyzed by a subclass B2 metallo-β-lactamase (CphA from Aeromonas hydrophila) is investigated using hybrid quantum mechanical/molecular mechanical methods and density functional theory. We focused on a recently proposed catalytic mechanism that involves a non-metal-binding water nucleophile in the active site of the monozinc CphA. Both theoretical models identified a single transition state featuring nearly concomitant nucleophilic addition and elimination steps, and the activation free energy from the potential of mean force calculations was estimated to be ∼14 kcal/mol. The theoretical results also identified the general base for activating the water nucleophile to be the metal-binding Asp-120 rather than His-118, as suggested earlier. The protonation of Asp-120 leads to cleavage of the Oδ2-Zn coordination bond, whereas the negatively charged nitrogen leaving group resulting from the ring opening replaces Asp-120 as the fourth ligand of the sole zinc ion. The electrophilic catalysis by the metal ion provides sufficient stabilization for the leaving group to avoid a tetrahedral intermediate. The theoretical studies provided detailed insights into the catalytic strategy of this unique metallo-β-lactamase. The initial nucleophilic substitution step of biapenem hydrolysis catalyzed by a subclass B2 metallo-β-lactamase (CphA from Aeromonas hydrophila) is investigated using hybrid quantum mechanical/molecular mechanical methods and density functional theory. We focused on a recently proposed catalytic mechanism that involves a non-metal-binding water nucleophile in the active site of the monozinc CphA. Both theoretical models identified a single transition state featuring nearly concomitant nucleophilic addition and elimination steps, and the activation free energy from the potential of mean force calculations was estimated to be ∼14 kcal/mol. The theoretical results also identified the general base for activating the water nucleophile to be the metal-binding Asp-120 rather than His-118, as suggested earlier. The protonation of Asp-120 leads to cleavage of the Oδ2-Zn coordination bond, whereas the negatively charged nitrogen leaving group resulting from the ring opening replaces Asp-120 as the fourth ligand of the sole zinc ion. The electrophilic catalysis by the metal ion provides sufficient stabilization for the leaving group to avoid a tetrahedral intermediate. The theoretical studies provided detailed insights into the catalytic strategy of this unique metallo-β-lactamase. The excessive use and abuse of β-lactam antibiotics have accelerated the spread of drug-resistant bacterial strains. The unprecedented level of antibiotic resistance threatens to destroy their efficacy in treating infectious diseases, posing a grand challenge to public health (1Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2373) Google Scholar). The primary defense strategy adopted by bacteria is to deactivate the antibiotics by hydrolytic cleavage of the β-lactam ring, catalyzed by β-lactamases (2Fisher J.F. Meroueh S.O. Mobashery S. Chem. Rev. 2005; 105: 395-424Crossref PubMed Scopus (760) Google Scholar). These enzymes can be divided into four classes (3Bush K. Jacoby G.A. Medeiros A.A. Antimicrob. Agents Chemother. 1995; 39: 1211-1233Crossref PubMed Scopus (2129) Google Scholar). Enzymes in classes A, C, and D utilize an active site serine in the covalent catalysis of the β-lactam hydrolysis, whereas class B consists of metalloenzymes with one or two zinc cofactors. Although the catalytic mechanism of the serine-based enzymes is relatively well established (2Fisher J.F. Meroueh S.O. Mobashery S. Chem. Rev. 2005; 105: 395-424Crossref PubMed Scopus (760) Google Scholar), our understanding of class B β-lactamases is less developed (4Wang Z. Fast W. Valentine A.M. Benkovic S.J. Curr. Opin. Chem. Biol. 1999; 3: 614-622Crossref PubMed Scopus (269) Google Scholar). Metallo-β-lactamases often have very broad substrate spectra (5Bush K. Clin. Infect. Dis. 1998; 27: S48-S53Crossref PubMed Scopus (204) Google Scholar) stemming apparently from the metal-dependent catalytic mechanism. Despite much effort, no clinically effective inhibitor has been found. On the other hand, increasing evidence in recent years has pointed to rapid proliferation of these metalloenzymes in pathogenic microorganisms (6Livermore D.M. Woodford N. Curr. Opin. Microbiol. 2000; 3: 489-495Crossref PubMed Scopus (310) Google Scholar). Hence, these enzymes represent a potentially more potent threat to the existing arsenal of β-lactam antibiotics than other classes of β-lactamases (5Bush K. Clin. Infect. Dis. 1998; 27: S48-S53Crossref PubMed Scopus (204) Google Scholar, 6Livermore D.M. Woodford N. Curr. Opin. Microbiol. 2000; 3: 489-495Crossref PubMed Scopus (310) Google Scholar). Class B β-lactamases can be further separated into three subclasses. Despite considerable sequence diversity (4Wang Z. Fast W. Valentine A.M. Benkovic S.J. Curr. Opin. Chem. Biol. 1999; 3: 614-622Crossref PubMed Scopus (269) Google Scholar), their catalytic scaffolds are relatively conserved. All class B β-lactamases have two potential metal binding sites (7Carfi A. Pares S. Duee E. Galleni M. Duez C. Frere J.-M. Dideberg O. EMBO J. 1995; 14: 4914-4921Crossref PubMed Scopus (404) Google Scholar, 8Concha N.O. Rasmussen B.A. Bush K. Herzberg O. Structure. 1996; 4: 823-836Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 9Fabiane S.M. Sohi M.K. Wan T. Payne D.J. Bateson J.H. Mitchell T. Sutton B.J. Biochemistry. 1998; 37: 12404-12411Crossref PubMed Scopus (216) Google Scholar, 10Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spencer J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (297) Google Scholar, 11Orellano E.G. Girardini J.E. Cricco J.A. Ceccarelli E.A. Vila A.J. Biochemistry. 1998; 37: 10173-10180Crossref PubMed Scopus (115) Google Scholar). Protein ligands in the so-called Zn1 site include three His residues in the B1 and B3 subclasses, but a His residue is replaced by Asn in B2 subclass β-lactamases. The Zn2 site has an Asp-Cys-His triad in the B1 and B2 subclasses, and the Cys residue is substituted by His in the B3 subclass. It is well established that B1 and B3 subclass β-lactamases are catalytically active with one zinc cofactor, typically in the Zn1 site (7Carfi A. Pares S. Duee E. Galleni M. Duez C. Frere J.-M. Dideberg O. EMBO J. 1995; 14: 4914-4921Crossref PubMed Scopus (404) Google Scholar), and the second zinc typically enhances the catalytic activity (11Orellano E.G. Girardini J.E. Cricco J.A. Ceccarelli E.A. Vila A.J. Biochemistry. 1998; 37: 10173-10180Crossref PubMed Scopus (115) Google Scholar, 12Wang Z. Benkovic S.J. J. Biol. Chem. 1998; 273: 22402-22408Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13de Seny D. Heinz U. Wommer S. Kiefer J.H. Meyer-Klaucke W. Galleni M. Frere J.-M. Bauer R. Adolph H.-W. J. Biol. Chem. 2001; 276: 45065-45078Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 14Rasia R.M. Vila A.J. Biochemistry. 2002; 41: 1853-1860Crossref PubMed Scopus (68) Google Scholar). However, the second zinc ion inhibits, rather than enhances, the catalytic activity of subclass B2 enzymes (15Valladares H.M. Felici A. Weber G. Adolph H.W. Zeppezauer M. Rossolini G.M. Amicosante G. Frere J.-M. Galleni M. Biochemistry. 1997; 36: 11534-11541Crossref PubMed Scopus (176) Google Scholar), which are only found in Aeromonas and have a strong preference toward carbapenems (6Livermore D.M. Woodford N. Curr. Opin. Microbiol. 2000; 3: 489-495Crossref PubMed Scopus (310) Google Scholar). Interestingly, the catalytic zinc cofactor in B2 β-lactamases occupies the Zn2 site, as demonstrated by recent experiments (15Valladares H.M. Felici A. Weber G. Adolph H.W. Zeppezauer M. Rossolini G.M. Amicosante G. Frere J.-M. Galleni M. Biochemistry. 1997; 36: 11534-11541Crossref PubMed Scopus (176) Google Scholar, 16Crawford P.A. Yang K.-W. Sharma N. Bennett B. Crowder M.W. Biochemistry. 2005; 44: 5168-5176Crossref PubMed Scopus (56) Google Scholar, 17Garau G. Bebrone C. Anne C. Galleni M. Frere J.-M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar). Until recently, it was generally believed that the nucleophile in the hydrolysis reaction catalyzed by class B β-lactamases is a metal-bound hydroxide (4Wang Z. Fast W. Valentine A.M. Benkovic S.J. Curr. Opin. Chem. Biol. 1999; 3: 614-622Crossref PubMed Scopus (269) Google Scholar). This hypothesis is supported by several x-ray structures of B1 and B3 β-lactamases, in which an oxygen moiety was observed to coordinate with one or both zinc ions (7Carfi A. Pares S. Duee E. Galleni M. Duez C. Frere J.-M. Dideberg O. EMBO J. 1995; 14: 4914-4921Crossref PubMed Scopus (404) Google Scholar, 8Concha N.O. Rasmussen B.A. Bush K. Herzberg O. Structure. 1996; 4: 823-836Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 9Fabiane S.M. Sohi M.K. Wan T. Payne D.J. Bateson J.H. Mitchell T. Sutton B.J. Biochemistry. 1998; 37: 12404-12411Crossref PubMed Scopus (216) Google Scholar, 10Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spencer J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (297) Google Scholar). However, a recent atomic resolution structure of a subclass B2 β-lactamase (CphA from Aeromonas hydrophila (15Valladares H.M. Felici A. Weber G. Adolph H.W. Zeppezauer M. Rossolini G.M. Amicosante G. Frere J.-M. Galleni M. Biochemistry. 1997; 36: 11534-11541Crossref PubMed Scopus (176) Google Scholar)) implicated a non-metal-binding active site water (17Garau G. Bebrone C. Anne C. Galleni M. Frere J.-M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar). Based on the structures of the enzyme in its native form and complexed with a hydrolysis intermediate, a mechanism advocating the nucleophilic role of this active site water was advanced by Garau et al. (17Garau G. Bebrone C. Anne C. Galleni M. Frere J.-M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar). This mechanism is very different from the conventional model based on a zinc-bound OH-. Given the unique sequence, substrate profile, and binding pattern of the sole zinc cofactor of subclass B2 β-lactamases, however, this alternative mechanism is not unreasonable but certainly requires more detailed investigations in both theoretical and experimental fronts. We report here a detailed theoretical investigation on the catalytic mechanism of the CphA enzyme in hydrolyzing a carbapenems antibiotic (biapenem). It dovetails our recent work on the Michaelis complex of the same system using a quantum mechanical/molecular mechanical (QM/MM) 2The abbreviations used are: QM/MM, quantum mechanical/molecular mechanical; DFT, density functional theory; SSC-DFTB, self-consistent charge density functional tight binding; B3LYP, Becke-3-Lee-Yang-Parr; PMF, potential of mean force; MD, molecular dynamics; TS, transition state; EI, enzyme-intermediate; ES, enzyme substrate. method and density functional theory (DFT) (18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar). We focus on the initial nucleophilic substitution step of the catalytic hydrolysis reaction and examine the putative nucleophilic role of the non-metal-binding water in the proposed mechanism using the same theoretical methods. DFT Model—The truncated active site includes the zinc ion and its three protein ligands His-263, Cys-221, and Asp-120, approximated respectively by an imidazole, a methyl thiolate, and an acetate. The substrate was approximated by a biapenem analog in which the bicycle-triazoliumthio group was replaced by -CH3. In addition, the model also includes an H2O and a methyl imidazole representing His-118. The Becke-3-Lee-Yang-Parr (B3LYP) exchange correlation functional and a standard basis set (6-31G**) were used. The geometries of stationary points were fully optimized with no geometric constraints, and the default convergence criterion was used. This is followed by the calculations of their harmonic vibrational frequencies and electrostatic potential charges. Finally, solvent effects were approximately taken into account with the polarized continuum model. Model studies of the hydrolysis of a biapenem analog catalyzed by two general bases, namely acetate and imidazole, were also performed at the B3LYP/6-31++G** level of theory. All DFT calculations reported in this work were performed using Gaussian 03. 3www.gaussian.com. QM/MM Models—A major disadvantage of the truncated active site model is its omission of the electrostatic and van der Waals microenvironment supplied by its surrounding protein residues and solvent. An approximate solution is to include the entire solvated protein but to partition the enzyme into a quantum mechanical region surrounded by a classical molecular mechanical region. This QM/MM approach (20Warshel A. Levitt M. J. Mol. Biol. 1976; 103: 227-249Crossref PubMed Scopus (3695) Google Scholar) has been quite successful in studying enzymatic reactions (21Gao J. Truhlar D.G. Annu. Rev. Phys. Chem. 2002; 53: 467-505Crossref PubMed Scopus (707) Google Scholar, 22Warshel A. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 425-443Crossref PubMed Scopus (443) Google Scholar). The advantages are particularly conspicuous for metalloenzymes, because the metal-ligand bonds are notoriously difficult to model with force fields. Following our earlier work (18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar), a QM/MM method is used to study the catalytic mechanism of the CphA enzyme. The model construction has been discussed in detail in our recent publication on the binding dynamics of CphA complexed with biapenem (18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar). Here, only a short description is given. Starting from the recent x-ray structure of the enzyme-intermediate complex (PDB code 1X8I) (17Garau G. Bebrone C. Anne C. Galleni M. Frere J.-M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar), the hydrolysis intermediate was removed, and hydrogens were added. The biapenem molecule was then manually docked at the enzyme active site in a manner consistent with the x-ray structures. After solvated in a pre-equilibrated sphere of TIP3P waters with a 25 Å radius, the enzyme-substrate complex was subjected to stochastic boundary conditions (23Brooks C.L. II I Karplus M. J. Mol. Biol. 1989; 208: 159-181Crossref PubMed Scopus (407) Google Scholar) designed to reduce computational costs by removing those atoms 25 Å away from the zinc ion and restricting the motion of atoms in the buffer zone (22 < r < 25 Å) using a Langevin model. Non-bonded interactions were cut off at 12 Å. The MM region in our simulations was characterized by the CHARMM all atom force field (24MacKerell Jr., A.D. Bashford D. Bellott M. Dunbrack Jr., R.L. Evanseck J.D. Field M.J. Fischer S. Gao J. Guo H. Ha S. Joseph-McCarthy D. Kuchnir L. Kuczera K. Lau F.T.K. Mattos C. Michnick S. Ngo T. Nguyen D.T. Prodhom B. Reiher III, W.E. Roux B. Schlenkrich M. Smith J.C. Stote R. Straub J. Watanabe M. Wiorkiewicz-Kuczera J. Yin D. Karplus M. J. Phys. Chem. B. 1998; 102: 3586-3616Crossref PubMed Scopus (11819) Google Scholar). The QM region contains Zn2+, its protein ligands Asp-120, Cys-221, and His-263, one crystal water (W11), and a potential general base His-118, as well as the biapenem substrate. The CHARMM van der Waals parameters were used for the 82 QM atoms. The interface between the QM and MM regions was approximated using link atoms (25Field M.J. Bash P.A. Karplus M. J. Comput. Chem. 1990; 11: 700-733Crossref Scopus (2185) Google Scholar), which were added to Cβ atoms of the four residues. All calculations reported here were carried out using CHARMM (26Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comput. Chem. 1983; 4: 187-217Crossref Scopus (14019) Google Scholar). In most of the calculations reported here, the QM part of the enzyme-substrate complex was treated with the self-consistent charge density functional tight binding (SCC-DFTB) method (27Elstner M. Porezag D. Jungnickel G. Elsner J. Haugk M. Frauenheim T. Suhai S. Seigert G. Phys. Rev. 1998; B58: 7260-7268Crossref Scopus (3322) Google Scholar) implemented in CHARMM (28Cui Q. Elstner M. Kaxiras E. Frauenheim T. Karplus M. J. Phys. Chem. B. 2001; 105: 569-585Crossref Scopus (561) Google Scholar). SCC-DFTB is an approximate but very efficient density functional theory. It has been extensively tested (28Cui Q. Elstner M. Kaxiras E. Frauenheim T. Karplus M. J. Phys. Chem. B. 2001; 105: 569-585Crossref Scopus (561) Google Scholar, 29Elstner M. Hobza P. Frauenheim T. Suhai S. J. Phys. Chem. B. 2001; 114: 5149-5155Crossref Scopus (951) Google Scholar) and applied successfully to several enzyme systems (30Riccardi, D., Schaefer, P., Yang, Y., Yu, H., Ghosh, N., Prat-Resina, X., Konig, P., Li, G., Xu, D., Guo, H., Elstener, M., and Cui, Q. (in press) J. Phys. Chem. BGoogle Scholar). A particularly important advance related to this work is the recent parameterization of biological zinc, which yielded results, such as geometry and ligand binding energies, which are in much better agreement with the B3LYP/6-311+G** model than other semiempirical methods (31Elstner M. Cui Q. Munih P. Kaxiras E. Frauenheim T. Karplus M. J. Comput. Chem. 2003; 24: 565-581Crossref PubMed Scopus (147) Google Scholar). To ensure the accuracy of SCC-DFTB/MM results, we have also performed single point B3LYP/CHARMM calculations along the semiempirical reaction path. The large size of the QM region forced us to employ a smaller basis set (6-31G) than that used in the truncated active site model. The reaction coordinate for the nucleophilic addition was defined as Rϕ = ROw-C7 (see Scheme 1 for atom definitions). This selection of the reaction coordinate does not specifically assign the general base, which can be either His-118 or Asp-120, as suggested by our previous simulations of the binding dynamics (18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar). In addition, this choice may also shed light on the nature of the reaction path. In particular, movement along the reaction coordinate would not lead to the enzyme-intermediate (EI) complex if a tetrahedral intermediate were to exist. The reaction path energy profile was determined by adiabatic mapping. The configurations obtained from the reaction path calculations were later used as the initial structures for the free energy simulation. In the potential of mean force (PMF) calculations, umbrella sampling (32Torrie G.M. Valleau J.P. J. Comput. Phys. 1977; 23: 187-199Crossref Scopus (4333) Google Scholar) with harmonic force constants in the 100∼180 kcal/mol·Å2 range was employed in twelve windows. The configuration space was sampled by classical molecular dynamics (MD). In each window, the minimal energy configuration was heated to 300 K in 30 ps followed by 30 ps of equilibration at the same temperature. The data were collected in the subsequent 40 ps. Finally, the PMF was obtained using the weighted histogram analysis method (WHAM) (33Kumar S. Bouzida D. Swendsen R.H. Kollman P.A. Rosenberg J.M. J. Comput. Chem. 1992; 13: 1011-1021Crossref Scopus (5045) Google Scholar). During the MD calculations, the SHAKE algorithm (34Ryckaert J.P. Ciccotti G. Berendsen H.J. J. Comput. Phys. 1977; 23: 327-341Crossref Scopus (17193) Google Scholar) was applied to maintain all covalent bonds involved hydrogen atoms, except for those in the catalytic water. The time step was 1 fs. DFT Model—As shown in our earlier work (18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar), the zinc binding site can be accurately described by a truncated active site model using DFT. Here, we have used a similar model to describe the nucleophilic substitution step of the hydrolysis reaction, with a larger basis set. The optimized ES complex in Fig. 1 was found to have a very similar geometry to that reported in Ref. 18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar. Some key geometric parameters are summarized in Table 1 along with average distances and angles obtained from our previous MD simulation of the enzyme-substrate complex. In particular, the zinc ion is tetracoordinated by four ligands, namely His-263, Cys-221, Asp-120, and the substrate carboxylate. A water molecule is located in a pocket formed by His-118, Asp-120, and the β-lactam ring, well positioned for the nucleophilic attack at the substrate carbonyl carbon (C7). The Ow···C7 distance is 3.38 Å, close to that observed in our earlier MD simulation (3.54 ± 0.58 Å). The water molecule forms hydrogen bonds with both Nδ1 of the His-118 imidazole ring and the metal-binding Oδ2 atom of Asp-120. The latter appears to be stronger than the former, as evidenced by the hydrogen bond distances of 2.43 (H···Nδ1) and 1.93 Å (H···Oδ2), respectively.TABLE 1Key geometric parameters of stationary points obtained at the B3LYP/6-31G** level of theory using the truncated active-site model and comparisons with QM/MM results The QM/MM data for the ES complex were taken from our earlier MD study (18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar) while those for the TS complex are from the reaction path calculations. The fluctuations in the MD results are also included.Truncated ModelQM/MMESTSEIESaData from MD simulations of the CphA-biapenem complex (18)TSbSaddle point geometry obtained by conjugate peaks refinement of the reaction pathDistance (A)N4···Zn2+3.422.211.953.38 ± 0.582.92O13···Zn2+1.932.051.952.12 ± 0.072.18Zn2+···Nϵ2(His-263)2.102.152.122.04 ± 0.072.02Zn2+···Oδ2(Asp-120)1.972.074.052.14 ± 0.072.14Zn2+···S(Cys-221)2.362.482.352.34 ± 0.082.27Ow···C73.381.701.373.54 ± 0.581.80N4···C71.411.813.112.04C7···O141.211.211.211.18H1···Oδ2(Asp-120)1.921.460.991.77H1···Ow0.981.051.801.02H2···Nδ1(His-118)2.432.011.851.55Bond angle (°)C7-N4-C3128.9112.0128.8124.4 ± 6.4141.4C2-S-C17101.2101.3101.4106.9 ± 3.5106.1O13···Zn2+···O†2(Asp-120)124.9134.2165.1124.2 ± 11.6133.3O13···Zn2+···S(Cys-221)107.496.0107.9118.3 ± 9.7108.2O13···Zn2+···Nϵ2(His-263)101.8108.8115.395.5 ± 5.092.7a Data from MD simulations of the CphA-biapenem complex (18Xu D. Zhou Y. Xie D. Guo H. J. Med. Chem. 2005; 48: 6679-6689Crossref PubMed Scopus (42) Google Scholar)b Saddle point geometry obtained by conjugate peaks refinement of the reaction path Open table in a new tab The existence of two basic side chains that are hydrogen-bonded with the nucleophilic water observed in both the DFT and QM/MM models raises an interesting question about the identity of the general base in the nucleophilic substitution step of the hydrolysis reaction. The mechanism proposed by Garau et al. (17Garau G. Bebrone C. Anne C. Galleni M. Frere J.-M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar) favors His-118, but the strong hydrogen bond with Asp-120 suggests an alternative with the carboxylate side chain group as the general base. To resolve this issue, we studied both scenarios within the truncated active site model by searching for reactive transition states. As displayed in Fig. 1, a transition state was located for the nucleophilic substitution step of the hydrolysis reaction with Asp-120 as the general base, and its optimized bond lengths and angles are also listed in Table 1. This first-order saddle point, as evidenced by a single imaginary frequency, features nearly concerted nucleophilic addition and elimination steps, as evidenced by a shortened Ow-C7 bond (1.68 versus 3.38 Å in the ES complex) and an elongated C7-N4 bond (1.86 versus 1.41 Å in the ES complex). The partial cleavage of the amide bond creates an anionic N4, evidenced by its negative charge (-0.45) at the transition state, in sharp contrast with its charge (0.06) in the reactant complex. As the water nucleophile approaches C7, one of its hydrogen atoms (H1) starts to move toward Oδ2 of Asp-120. The Ow···H1 and H1···Oδ2 distances at the transition state are 1.05 and 1.50 Å, respectively. As a result, the metal-ligand bond between the Asp-120 carboxylate group and zinc ion becomes slightly impaired as the distance Oδ2···Zn increases from 1.97 to 2.08 Å. In the meantime, the negative charge buildup at N4 due to the C-N bond cleavage provides incentives for it to move toward the zinc ion. The corresponding N4···Zn distance at the transition state becomes 2.21 from 3.44 Å in the ES complex. The zinc ion is pentacoordinated in the transition state. No tetrahedral intermediate was found in this reaction pathway, and the intrinsic reaction coordinate leads directly to the ES complex in one direction and the EI complex in the other. The EI complex is displayed in Fig. 1, and selected bond lengths and angles are listed in Table 1 along with those for other stationary points. The procession from the transition state to the EI complex results in the complete cleavage of the lactam amide bond, accompanied by a concomitant proton transfer to the metal-binding Asp-120. The neutralization of the Asp-120 side chain renders it an ineffective ligand to the zinc ion, leading to a final Oδ2···Zn distance of 4.10 Å, signifying the breaking of the bond. In the meantime, the negatively charged N4 (q = -0.70) replaces Asp-120 as the fourth ligand to the zinc ion, as evidenced by the short (1.95 Å) N4···Zn distance. The resulting EI complex can be converted to the enzyme product complex by proton transfer and possibly intramolecular rearrangements, as suggested by the recent x-ray structure (17Garau G. Bebrone C. Anne C. Galleni M. Frere J.-M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar). However, the latter processes were not studied here. The energies, zero-point corrected energies, and free energies of all of the stationary points are summarized in Table 2. The reaction barrier and exothermicity are 31.7 and 1.1 kcal/mol, respectively, at the B3LYP/6-31G** level. The basis set effect seems to be small as the corresponding values at the B3LYP/6-31G* are 32.0 and 1.2 kcal/mol, respectively. The activation free energy is estimated to be 35.0 kcal/mol. The relatively large reaction barrier is likely because of the lack of the protein/solvent environment in the truncated model. After the solvent effects are included using the polarized continuum model, for example, the barrier is reduced by ∼6 kcal/mol.TABLE 2Energetics (kcal/mol) for the nucleophilic substitution step of the hydrolysis reaction The truncated active site model at the B3LYP/6-31G** level of theory was used.EnergeticsaAll energies are given relative to ESTSEIEnergy31.71.1Energy with ZPE correction31.22.2Free energy35.02.4Free energy (PCM)bAqueous solution (ϵ = 80)29.10.6a All energies are given relative to ESb Aqueous solution (ϵ = 80) Open table in a new tab However, repeated efforts to locate the transition state with His-118 as the general base failed. To understand the capacity of the His or Asp residue as a general base in catalyzing the hydrolysis of β-lactams, we examined simple model systems without the metal ion and its protein ligands in which the two amino acid side chains are approximated by an imidazole ring and acetate, respectively. Five stationary states were located for the acetate-catalyzed reaction, including the reactant complex, transition state for nucleophilic addition (TS1), tetrahedral intermediate, transition state for elimination (TS2), and product complex. They are displayed in Fig. 2 with key bond lengths indicated in the figure. The rate-limiting step is the formation of the tetrahedral intermediate, whereas the barrier for elimination is very small. These results are consistent with previous theoretical studies of β-lactam hydrolysis (35Coll M. Frau J. Vilanova B. Donoso J. Munoz F. Blanco F.G. J. Phys. Chem. B. 2000; 104: 11389-11394Crossref Scopus (22) Google Scholar, 36Diaz N. Suarez A. Sordo J.A. Tunon I. Silla E. Chem. Eur. J. 2002; 8: 859Crossref PubMed Scopus (15) Google Scholar). When the imidazole group was used as the general base, on the other hand, no reactive transition state was located after an exhaustive search. Reaction path calculations showed a monotonically increased energy profile in the nucleophilic addition coordinate. These results indicate that Asp is a better catalyst than His in the gas phase. This issue will be further addressed below with a QM/MM model in which the protein/solvent environment is included in the simulations. QM/MM Models—Although the truncated active site model provides insightful information about the catalytic reaction, it includes neither the protein/solvent environment nor the corresponding dynamics. To bridge the gap, we investigated the same nucleophilic substitution step of the β-lactam hydrolysis reaction employing two QM/MM models, namely the SCC-DFTB/CHARMM and B3LYP/CHARMM models. The minimal energy profile along the reaction coordinate was first determined by adiabatic mapping. The geometric parameters of the transition state, which was obtained by conjugate peak refinement (37Fischer S. Karplus M. Chem. Phys. Lett. 1992; 194: 252-261Crossref Scopus (426) Google Scholar), are listed in Table 1, and their agreement with the DFT transition state is quite reasonable, although imperfect. As in
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