Ab initio QM/MM dynamics simulation of the tetrahedral intermediate of serine proteases: insights into the active site hydrogen-bonding network

Ab initio QM/MM dynamics simulation is employed to examine the stability of the tetrahedral intermediate during the deacylation step in elastase-catalyzed hydrolysis of a simple peptide. An extended quantum region includes the catalytic triad, the tetrahedral structure, and the oxyanion hole. The calculations indicate that the tetrahedral intermediate of serine proteases is a stable species on the picosecond time scale. On the basis of geometrical and dynamical properties, and in agreement with many experimental and theoretical studies, it is suggested that the crucial hydrogen bonds involved in stabilizing this intermediate are between Asp-102 and His-57 and between the charged oxygen of the intermediate and the backbone N-H group of Gly-193 in the oxyanion hole. The mobility of the imidazolium ring between O(w) and O(gamma), two of the oxygens of the tetrahedral structure, shows how the intermediate could proceed toward the product state without a "ring-flip mechanism", proposed earlier on the basis of NMR data. In addition to the proposed C(epsilon)(1)-H.O hydrogen bond between the imidazolium ring and the backbone carbonyl of Ser-214, we observe an alternative C(epsilon)(1)-H.O hydrogen bond with the backbone carbonyl of Thr-213, that can stabilize the intermediate during the imidazolium movement. Proton hopping occurs between Asp-102 and His-57 during the simulation. The proton is, however, largely localized on the nitrogen, and hence it does not participate in a low-barrier hydrogen bond. The study also suggests factors that may be implicated in product release: breaking the hydrogen bond of the charged oxygen with the backbone of Ser-195 in the oxyanion hole and a loop opening between residues 216-225 that enables the breaking of a hydrogen bond in subsite S(3).     

Ab initio and density functional theory studies of the catalytic mechanism for ester hydrolysis in serine hydrolases

We present results from ab initio and density functional theory studies of the mechanism for serine hydrolase catalyzed ester hydrolysis. A model system containing both the catalytic triad and the oxyanion hole was studied. The catalytic triad was represented by formate anion, imidazole, and methanol. The oxyanion hole was represented by two water molecules. Methyl formate was used as the substrate. In the acylation step, our computations show that the cooperation of the Asp group and oxyanion hydrogen bonds is capable of lowering the activation barrier by about 15 kcal/mol. The transition state leading to the first tetrahedral intermediate in the acylation step is rate limiting with an activation barrier (ΔE₀) of 13.4 kcal/mol. The activation barrier in the deacylation step is smaller. The double-proton-transfer mechanism is energetically unfavorable by about 2 kcal/mol. The bonds between the Asp group and the His group, and the hydrogen bonds in the oxyanion hole, increase in strength going from the Michaelis complex toward the transition state and the tetrahedral intermediate. In the acylation step, the tetrahedral intermediate is a very shallow minimum on the energy surface and is not viable when molecular vibrations are included. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 69: 89–103, 1998     

Experimental evidence for enzyme-enhanced coupled motion/quantum mechanical hydrogen tunneling by ketosteroid isomerase

Although there are considerable data demonstrating that quantum mechanical hydrogen tunneling (HT) occurs in both enzymatic and nonenzymatic systems, little data exist that address the question of whether enzymes enhance the amount of HT relative to the corresponding nonenzymatic reactions. To investigate whether 3-oxo-Delta (5)-steroid isomerase (ketosteroid isomerase, KSI) enhances HT relative to the nonenzymatic (acetate-catalyzed) isomerization of Delta (5)-androstene-3,17-dione ( 1) to Delta (4)-androstene-3,17-dione ( 3), alpha-secondary deuterium kinetic isotope effects (KIE) at C-6 of the steroid were determined for both the KSI- and acetate-catalyzed isomerizations. The normal intrinsic secondary KIE for both wild type (WT) KSI (1.073 +/- 0.023) and acetate (1.031 +/- 0.010) suggest the possibility of coupled motion (CM)/HT in both the enzymatic and nonenzymatic systems. To assess the contribution of CM/HT in these reactions, the secondary KIE were also measured under conditions in which deuterium instead of hydrogen is transferred. The decrease in secondary KIE for WT (1.035 +/- 0.011) indicates the presence of CM/HT in the enzymatic reaction, whereas the acetate reaction shows no change in secondary KIE for deuterium transfer (1.030 +/- 0.009) and therefore no evidence for CM/HT. On the basis of these experiments, we propose that KSI enhances the CM/HT contribution to the rate acceleration over the solution reaction. Active site mutants of KSI (Y14F and D99A) yield secondary KIEs similar to that of WT, indicating that mutations at the hydrogen-bonding residues do not significantly decrease the contribution of CM/HT to the KSI reaction.     

Analysis of catalytic mechanism of serine proteases. Viability of the ring-flip hypothesis

Quantum calculations are applied to the active site of serine proteases, including four specific residues and a water molecule, as well as a substrate and proton donors in the oxyanion hole. Residues are tethered to the protein backbone of an X-ray structure but otherwise allowed to move freely to their lowest energy positions. The viability of the ring-flip hypothesis, which proposes that a 180 degrees rotation of the His-57 imidazole ring facilitates the catalysis, is assessed by comparison of energies of configurations both before and after such a flip. Specifically considered is the contribution to catalysis of the Ser-214 residue and a water molecule that is observed in the active site. The calculations provide detailed information concerning the nature, geometry, and strength of hydrogen bonds that are formed within the active site at each stage of the enzymatic mechanism.     

Testing geometrical discrimination within an enzyme active site: constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole

Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 A scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2-1.5 A resolution X-ray crystallography, (1)H and (19)F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.     

Conformational switching between carboxylic acid and carboxylate anion states by NH-O hydrogen bonding

Biologically important Ca-proteins and Ca-biominerals as metal-polymer complexes are often regulated by the complexation and demetalation with the biopolymers. Metal-oxygen bond is supported by NH···O hydrogen bonds between coordinating oxyanion and neighboring amide NH and by the successive hydrogen bonding networks. Carboxylate anion under hydrophobic conditions has a high basicity that leads to a covalent Ca-O bond character that is significantly affected by the NH···O hydrogen bond. The hydrogen bonds in Asp-containing tripeptide fragments in Ca-proteins presumably control coordination/dissociation of metal-oxygen bonds. Our systematic studies of carboxylate, sulfonate and phosphate Ca(II) complexes demonstrate a relationship between the basicity of oxyanion in carboxylate and hydrogen bonds as cooperating with the oligopeptide conformation in Asp-containing Ca(II) complexes. Hydrogen bonds between carboxylate oxyanion and amide NH, controlled by a conformational switching of oligopeptide fragments, seem to be one of essential factors for the regulatory formation of Ca-proteins and nano-architectures in connection with the interface structure of inorganic and organic phases in biominerals.     

Spectrochemical, ab initio and density functional studies on the conversion of 2-hydroxybenzonitrile (o-cyanophenol) into the oxyanion

The spectral and structural changes caused by the conversion of 2-hydroxybenzonitrile (o-cyanophenol) into the corresponding oxyanion have been followed by IR spectra, ab initio and density functional force field calculations. In agreement between theory and experiment, the conversion is accompanied by a 29 cm(-1) frequency decrease of the cyano stretching band, 2.7-fold increase in its integrated intensity, 5.8-fold (total value) intensification of the aromatic skeletal bands of Wilson's 8 and 19 types, and other essential spectral changes. According to the calculations, the strongest structural changes are the shortening of the Ph-O bond with 0.10 A, lengthenings of the adjacent CC bonds in the phenylene ring with 0.06 A and bond angle variations near the oxyanionic center. All these changes are connected with the formation of a quasi-ortho-quinonoidal structure of the o-phenylene ring in the oxyanion. According to the electronic density analysis, 0.41 e(-) (Mulliken) or 0.56 e(-) (natural bond orbital, NBO) of the anionic charge remain localized at the oxyanionic center. Conformations and hydrogen bonds have also been discussed on the basis of experimental and theoretical data.     

Contribution of the intramolecular hydrogen bond to the shift of the pKa value and the oxidation potential of phenols and phenolate anions

Intramolecularly OHO[double bond, length as m-dash]C hydrogen bonded phenols, 2-HO-C6H2-3,5-(t-Bu)2-CONH-t-Bu (1-OH), 2-HO-C6H2-5-t-Bu-1,3-(CONH-t-Bu)2 (2-OH) and 2-HO-C6H2-3,5-(t-Bu)2-NHCO-t-Bu (4-OH), were synthesized and their phenolate anions were prepared as tetraethylammonium salts (-1O-(NEt4+), 2-O-(NEt4+) and 4-O-(NEt4+)) with intramolecular NHO(oxyanion) hydrogen bonds. 4-HO-C(6)H(2)-3,5-t-Bu(2)-CONH-t-Bu (3-OH) and its phenolate anion, 3-O-(NEt4+), were synthesized as non-hydrogen bonded references. The presence of intramolecular hydrogen bonds was established through the crystallographic analysis and/or (1)H NMR spectroscopic results. Intramolecular NHO(phenol) hydrogen bonds shift the pK(a) of the phenol to a more acidic value. The results of cyclic voltammetry show that the intramolecular OH...O=C hydrogen bond negatively shifts the oxidation potential of the phenol. In contrast, the intramolecular NHO(oxyanion) hydrogen bond positively shifts the oxidation potential of the phenolate anion, preventing oxidation. These contributions of the hydrogen bond to the pKa value and the oxidation potentials probably play an important role in the formation of a tyrosyl radical in photosystem II.     

Effect of electrostatic interaction on the mechanism of dehalogenation catalyzed by haloalkane dehalogenase

Theoretical calculation has been carried out for the nucleophilic displacement reaction of 1,2‐dichloroethane catalyzed by haloalkane dehalogenase. The results indicate that different hydrogen bond patterns of the oxyanion hole and the halide‐stabilizing residues play an important role in the dehalogenation reaction. They cause concertedly an earlier transition state (TS) with the activation barrier of 16.60 kcal/mol. The stabilization effect of Trp125 and Trp175 on chlorine atom in the TS is larger than that of the reactant complex by 15.67 kcal/mol so that, they make contribution to the stabilization of the TS. Moreover, the reaction shows the enzymatic action can be attributed to a combination of reactant‐state destabilization and transition‐state electrostatic stabilization. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Contribution of multicentered short hydrogen bond arrays to potency of active site-directed serine protease inhibitors

We describe and compare the pH dependencies of the potencies and of the bound structures of two inhibitor isosteres that form multicentered short hydrogen bond arrays at the active sites of trypsin, thrombin, and urokinase type plasminogen activator (urokinase or uPA) over certain ranges of pH. Depending on the pH, short hydrogen bond arrays at the active site are mediated by two waters, one in the oxyanion hole (H(2)O(oxy)) and one on the other (S2) side of the inhibitor (H(2)O(S2)), by one water (H(2)O(oxy)), or by no water. The dramatic variation in the length of the active site hydrogen bonds as a function of pH, of inhibitor, and of enzyme, along with the involvement or absence of ordered water, produces a large structural manifold of active site hydrogen bond motifs. Diverse examples of multicentered and two-centered short hydrogen bond arrays, both at and away from the active site, recently discovered in several protein crystal systems, suggest that short hydrogen bonds in proteins may be more common than has been recognized. The short hydrogen bond arrays resemble one another with respect to ionic nature, highly polar environment, multitude of associated ordinary hydrogen bonds, and disparate pK(a) values of participating groups. Comparison of structures and K(i) values of trypsin complexes at pH values where the multicentered short hydrogen bond arrays mediating inhibitor binding are present or absent indicate that these arrays have a minor effect on inhibitor potency. These features suggest little covalent nature within the short hydrogen bonds, despite their extraordinary shortness (as short as 2.0 A).     

Designing Foldamer–Foldamer Interactions in Solution: The Roles of Helix Length and Terminus Functionality in Promoting the Self‐Association of Aminoisobutyric Acid Oligomers

The biological activity of antibiotic peptaibols has been linked to their ability to aggregate, but the structure–activity relationship for aggregation is not well understood. Herein, we report a systematic study of a class of synthetic helical oligomer (foldamer) composed of aminoisobutyric acid (Aib) residues, which mimic the folding behavior of peptaibols. NMR spectroscopic analysis was used to quantify the dimerization constants in solution, which showed hydrogen‐bond donors at the N terminus promoted aggregation more effectively than similar modifications at the C terminus. Elongation of the peptide chain also favored aggregation. The geometry of aggregation in solution was investigated by means of titrations with [D₆]DMSO and 2D NOE NMR spectroscopy, which allowed the NH protons most involved in intermolecular hydrogen bonds in solution to be identified. X‐ray crystallography studies of two oligomers allowed a comparison of the inter‐ and intramolecular hydrogen‐bonding interactions in the solid state and in solution and gave further insight into the geometry of foldamer–foldamer interactions. These solution‐based and solid‐state studies indicated that the preferred geometry for aggregation is through head‐to‐tail interactions between the N and C termini of adjacent Aib oligomers.     

Calculation of relative binding affinities of fructose 1,6-bisphosphatase mutants with adenosine monophosphate using free energy perturbation method

The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between inhibitors and protein variants. In this article, the importance of hydrophobic and hydrophilic residues to the binding of adenosine monophosphate (AMP) to the fructose 1,6-bisphosphatase (FBPase), a target enzyme for type-II diabetes, was examined by FEP method. Five mutations were made to the FBPase enzyme with AMP inhibitor bound: 113Tyr --> 113Phe, 31Thr --> 31Ala, 31Thr --> 31Ser, 177Met --> 177Ala, and 30Leu --> 30Phe. These mutations test the strength of hydrogen bonds and van der Waals interactions between the ligand and enzyme. The calculated relative free energies indicated that: 113Tyr and 31Thr play an important role, each via two hydrogen bonds affecting the binding affinity of inhibitor AMP to FBPase, and any changes in these hydrogen bonds due to mutations on the protein will have significant effect on the binding affinity of AMP to FBPase, consistent to experimental results. Also, the free energy calculations clearly show that the hydrophilic interactions are more important than the hydrophobic interactions of the binding pocket of FBPase.     

Role of the catalytic triad and oxyanion hole in acetylcholinesterase catalysis: an ab initio QM/MM study

The initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE) has been studied by a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach. The reaction proceeds through the nucleophilic addition of the Ser203 O to the carbonyl C of acetylcholine, and the reaction is facilitated by simultaneous proton transfer from Ser203 to His447. The calculated potential energy barrier at the MP2(6-31+G) QM/MM level is 10.5 kcal/mol, consistent with the experimental reaction rate. The third residue of the catalytic triad, Glu334, is found to be essential in stabilizing the transition state through electrostatic interactions. The oxyanion hole, formed by peptidic NH groups from Gly121, Gly122, and Ala204, is also found to play an important role in catalysis. Our calculations indicate that, in the AChE-ACh Michaelis complex, only two hydrogen bonds are formed between the carbonyl oxygen of ACh and the peptidic NH groups of Gly121 and Gly122. As the reaction proceeds, the distance between the carbonyl oxygen of ACh and NH group of Ala204 becomes smaller, and the third hydrogen bond is formed both in the transition state and in the tetrahedral intermediate.     

A molecular dynamics investigation on the interaction properties of AzrC and its cofactor

In this report, the main contributions of FMN were employed in the reductive cleavage reaction of AzrC protein (as a member of azoreductase family). Molecular dynamics simulations of three models in the presence and absence of FMN and ligand were performed to gather information about the dynamic nature of active site residues of AzrC. Combination of pairwise decomposition and alanine scanning calculations provides critical information about the FMN binding sites. The MD results analyzed by alanine scanning method revealed the high negative scores for N 10 (A) A, N 12 (A) A, S 17 (A) A and Y 151 (A) A mutations, which were in agreement with pairwise decomposition analyses. Hydrogen bond analyses indicated that these residues play critical roles in establishing appropriate hydrogen bonds between AzrC and FMN. Negative energy results for nonpolar residues such as W 103 (A), M 102 (A) and F 105 (A) and binding free energy analyses of three complexes indicate that the VDW interactions could be regarded as some favorable contribution in FMN and AzrC protein and confirmed the critical role of FMN in ligand binding (35.84 %), in addition to its catalytic function. This information could be used for future experimental investigations.     

An Adjustable Cleft Based on an 8-Sulfonamide-2-Naphthoic Acid with Oxyanion Hole Geometry

Cleft type receptors showing the oxyanion hole motif have been prepared in a straightforward synthesis starting from the commercial 3,7-dihidroxy-2-naphthoic acid. The double H-bond donor pattern is achieved by the introduction of a sulfonamide group in the C-8 position of naphthalene and a carboxamide at the C-2 position. This cleft, for which the geometry resembles that of an oxyanion hole, is able to adjust to different guests, as shown by the analysis of the X-ray crystal structures of associates with methanol or acetic acid. Combination of hydrogen bonds and charge-transfer interactions led to further stabilization of the complexes, in which the electron-rich aromatic ring of the receptor was close in space to the electron-deficient dinitroaromatic guests. Modelling studies and bidimensional NMR experiments have been carried out to provide additional information.     

Molecular dynamics and quantum chemical studies on the catalytic mechanism of Delta5-3-ketosteroid isomerase: the catalytic diad versus the cooperative hydrogen bond mechanism

To further understand Delta(5)-3-ketosteroid isomerase (KSI) catalysis, we carried out molecular dynamics (MD) simulations of the KSI dimer ligated with a substrate and reaction intermediate analogue and high level ab initio calculations on relevant enzymatic reaction models. Simulation of the enzyme-substrate complex dimer systems showed asymmetric dynamics between the two monomers, in which the hydrogen bond pattern between the substrate and active site residues in the first and the second subunits supported the cooperative hydrogen bond (CH) and the catalytic diad (CD) mechanisms, respectively. On the other hand, only the CH mechanism was supported in the MD simulation of the enzyme-intermediate complex dimer. From MP2/6-31+G**//RHF/6-31G** calculations, we found the kinetic barriers for the two reaction mechanisms were similar. The CH route afforded a greater stabilization to the enolate intermediate than did the CD counterpart. Thus, the present computational studies indicate that the CH mechanism would be favored over the CD one in the catalytic action of KSI. However, the latter could not be ruled out conclusively because of the explicit appearance of a CD configuration in the MD trajectories of the enzyme-substrate complex and because of the similar intrinsic activation barrier for the CH and CD mechanisms. The appearance of configurations that favor the CD pathway is rationalized in terms of a model in which the KSI-substrate complex does not have a strong preference for one hydrogen bonding pattern over another, while the KSI-intermediate complex favors a cooperative hydrogen bond pattern in order to stabilize the reaction intermediate. This hypothesis is supported by the ab initio calculations which indicate that the CH intermediate is more stable than the CD one by approximately 6.3 kcal/mol.     

Neutral and ionic hydrogen bonding in Schiff bases

Low-temperature, high-resolution X-ray studies of charge distributions in the three Schiff bases, the dianil of 2-hydroxy-5-methylisophthaldehyde, 3,5-dinitro-N-salicylidenoethylamine and 3-nitro-N-salicylidenocyclohexylamine, have been carried out. These structures exhibit interesting weak interactions, including two extreme cases of intramolecular hydrogen bonds that are ionic N(+)-H...O- and neutral O-H...N in nature. These two types of hydrogen bond reflect differences in geometrical parameters and electron density distribution. At the level of geometry, the neutral O-H...N hydrogen bond is accompanied by an increase in the length of the C(1)-O(1) bond, opening of the ipso-C(1) angle, elongation of the aromatic C-C bonds, shortening of the C(7)-N(2) bond and increased length of the C(1)-C(7) bond, relative to the ionic hydrogen bond type. According to the geometrical and critical point parameters, the neutral O-H...N hydrogen bond seems to be stronger than the ionic ones. There are also differences between charge density parameters of the aromatic rings consistent with the neutral hydrogen bond being stronger than the ionic ones, with a concomitant reduction in the aromaticity of the ring. Compounds with the ionic hydrogen bonds show a larger double-bond character in the C-O bond than appears in the compound containing a neutral hydrogen bond; this suggests that the electronic structure of the former pair of compounds includes a contribution from a zwitterionic canonical form. Furthermore, in the case of ionic hydrogen bonds, the corresponding interaction lines appear to be curved in the vicinity of the hydrogen atoms. In the 3-nitro-N-salicylidenocyclohexylamine crystal there exists, in addition to the intramolecular hydrogen bond, a pair of intermolecular O...H interactions in a centrosymmetric dimer unit.     

Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K

Molecular dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have determined the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aqueous solution. The free energy of activation for the deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aqueous solution. The reduction of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen-bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 A and placing the oxyanion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and for the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton-transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.     

A theoretical study of the hydrogen bond donor capability and co-operative effects in the hydrogen bond complexes of the diaza-aromatic betacarbolines

In this work, we present a quantum mechanical investigation on the hydrogen bond interactions of N(9)-methyl-9H-pyrido[3,4-b]indole, MBC, and N(2)-methyl-9H-pyrido[3,4-b]indole, BCA, with different hydrogen bond donors. Thus, it has been analysed the influence that the hydrogen bond donor strength and the co-operative effect of the increasing number of donor molecules have on the shape of the potential energy surfaces versus the N···H distances, r(N–H). To rationalize the nature of the interactions, the Bader theory has been applied and the characteristics of the bond critical points analysed. The results show that two different hydrogen bond complexes can be formed depending on the donor capabilities or the number of donor molecules included in the calculations. The topological parameters from the Bader theory are used to justify the statement that the analysed interactions can be classified as weak or partially covalent hydrogen bond interactions, respectively. As experimentally observed, weak hydrogen bond donors form weak hydrogen bond complexes, called HBC. Upon the increase of the donor strength the N···H proton is shifted nearest to the nitrogen atom giving rise to the observation of a stronger hydrogen bond complex, the proton transfer complex, PTC. The most outstanding result of these studies is the fact that the formation of the PTC can also be managed just by changing the number of donor molecules, that is, by a co-operative effect of the hydrogen bonds.     

The Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological Dehalogenation

The biological dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole‐enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen‐bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the α‐carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C? F bond is greatly facilitated by the hydrogen‐bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.