Non-Enzymatic and Enzymatic Hydrolysis of Alkyl Halides

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 8417–8420, August 1997 Biochemistry

Non-enzymatic and enzymatic hydrolysis of alkyl halides: A haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an SN2 displacement reaction

FELICE C. LIGHTSTONE,YA-JUN ZHENG,ANDREAS H. MAULITZ, AND THOMAS C. BRUICE*

Department of Chemistry, University of California, Santa Barbara, CA 93106

Contributed by Thomas C. Bruice, May 23, 1997

ABSTRACT The semiempirical PM3 method, calibrated reactions with the haloalkane dehalogenase of Xanthobacter .(against ab initio HF͞6–31؉G(d) theory, has been used to autothropicus meet these criteria (8 elucidate the reaction of 1,2-dichloroethane (DCE) with the Dehalogenation enzymes have attracted considerable atten- carboxylate of Asp-124 at the active site of haloalkane deha- tion owning to the potential of applying these enzymes to the logenase of Xanthobacter autothropicus. Asp-124 and 13 other treatment of halogenated hydrocarbon-contaminated soil and amino acid side chains that make up the active site cavity water supplies (9–11). Haloalkane dehalogenase is of partic- (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val- ular interest because it catalyzes hydrolysis of alkyl halides 219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His- without requiring any cofactors or metal ions (8). The enzy- 289) were included in the calculations. The three most signif- matic hydrolysis of alkyl halides to the corresponding alcohols icant observations of the present study are that: (i) the DCE follows a two-step process, which involves the formation of an substrate and Asp-124 carboxylate, in the reactive ES com- alkyl-enzyme ester intermediate (12–14). Our interest is in the plex, are present as an ion-molecule complex with a structure initial step of the enzymatic-dehalogenation reaction, which similar to that seen in the gas-phase reaction of AcO؊ with involves a nucleophilic attack of the carboxylate group of DCE; (ii) the structures of the transition states in the gas- Asp-124 on the halogen-bearing carbon, displacing the chloride via an S 2-displacement reaction (Scheme1). From ex- phase and enzymatic reaction are much the same where the N amination of the x-ray crystallographic structure of the ES structure formed at the active site is somewhat exploded; and complex, Verschueren et al. (12–14) observed the indole NH (iii) the enthalpies in going from ground states to transition groups of Trp-125 and Trp-175 to be in position to hydrogen states in the enzymatic and gas-phase reactions differ by only bond to the leaving chloride and proposed this to be assisting a couple kcal͞mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorga- nization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

For a catalytic reaction, knowledge of the structure of the critical transition state (TS) tells much about the means of catalysis. This is particularly so in the case of enzyme catalysis, considering the school of thought that enzyme catalysis is due to transition-state stabilization (1–4). Even though experimental techniques such as x-ray diffraction and NMR have pro- vided and will continue to provide valuable structural infor- mation concerning the enzyme–substrate (ES), enzyme– intermediate, and enzyme–product complexes, it is highly unlikely that experimental techniques will ever allow direct observation of the transition state in an enzymatic reaction SCHEME 1 because of the extremely short life time of the transition state. in departure of ClϪ from the haloalkane. This feature has since An approach to characterizing a TS at the active site of an been confirmed by other experimental studies (15). The enzyme is to use quantum mechanical theory (5–7). To test this simplicity of the bimolecular S 2 displacement of ClϪ from a procedure, the following criteria must be met. First, for the N primary haloalkane by a carboxylate anion allows the com- theoretical results to be meaningful, the method should be able parison of the transition states for such a displacement in to treat at least the amino acid residues that line the active site non-enzymatic and enzymatic reactions. Using ab initio and of the enzyme. Second, the enzyme and the substrate must be semiempirical molecular orbital theory, the non-enzymatic small in size. Third, for the sake of simplicity, the reaction reaction of Eq. 1 has recently been studied in both gas phase being studied should consist of a single step, thereby obviating and in solution in this laboratory (16). Here we report the intermediates. Fourth, a well studied enzyme must be chosen results of an investigation of the enzymatic reaction. so that the theoretical results can be validated. The catalytic Ϫ Ϫ CH3CO2 ϩ ClCH2CH2Cl 3 CH3CO2OCH2CH2Cl ϩ Cl [1] The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: ES, enzyme substrate; TS, transition state; DCE, accordance with 18 U.S.C. §1734 solely to indicate this fact. 1,2-dichloroethane. © 1997 by The National Academy of Sciences 0027-8424͞97͞948417-4$2.00͞0 *To whom reprint requests should be addressed. e-mail: PNAS is available online at http:͞͞www.pnas.org. [email protected].

8417 Downloaded by guest on September 28, 2021 8418 Biochemistry: Lightstone et al. Proc. Natl. Acad. Sci. USA 94 (1997)

Currently, it is computationally prohibitive to include an entire energy minimum, whereas the x-ray crystal structure is a enzyme in a quantum mechanical computation. In the present time-averaged structure. The calculated ground state is very study, the active site model consists of a crystallographic water similar to the gas-phase ion–molecule complex for the non- and the 14 amino acid residues (Glu-56, Asp-124, Trp-125, enzymatic reaction (Eq. 1) of DCE with acetate (16). It should Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro- be noted that in 2, DCE is in a gauche-like conformation with 223, Val-226, Leu-262, Leu-263, and His-289; see Fig. 11) that a Cl-C-C-Cl dihedral angle of 74.2°, which also is similar to the surround the Asp-124 carboxylate anion nucleophile and the dihedral angle in the gas-phase ion–molecule complex (16). In 1,2-dichloroethane (DCE) substrate. Additional hydrogen at- the original x-ray crystallographic study, DCE was built into oms were included to satisfy all valences of the crystal structure the enzyme active site in a trans conformation during structure coordinates; the resulting system of partial amino acid struc- refinement, although the electron density for one of the tures and substrate contains 288 atoms. To retain the overall chlorines is not very strong (12–14). However, as indicated by structure of the system during the calculations, the peptide our previous ab initio calculations on the non-enzymatic backbone atoms were held fixed to their x-ray crystallographic reaction of AcOϪ with DCE and the current calculations in the coordinates (2dhc) to compensate for the absence of the rest active site, DCE is probably in a gauche conformation or a of the enzyme. All side chain atoms and DCE were allowed to mixture of both trans and gauche when bound in the active site move. The PM3 (17) hamiltonian as implemented in GAUSSIAN of the enzyme. The gauche form is expected to have a stronger 94 (18) was used. PM3 was chosen based on the following interaction with the enzyme than the trans form because the observations. Our previous study (16) has demonstrated that former has a dipole. If DCE is in the trans form on approach the semiempirical PM3 method provides essentially the same to the carboxylate of Asp-124, there will be a large repulsion transition-state structure for the reaction of Eq. 1 as does ab between the negatively charged carboxylate group and the initio HF͞6–31ϩG(d) level of theory. The semiempirical PM3 chlorine on the adjacent carbon of DCE. This repulsion gets was chosen over AM1 (19) for comparison with ab initio stronger as DCE gets closer to the carboxylate; a simple calculations because AM1 tends to give bifurcated hydrogen conformational change (trans to gauche) alleviates this unfa- bonding geometries (20). Also, the large error associated with vorable interaction. Thus, the active form of DCE is the gauche AM1-calculated heat of formation of chloride ion (21) further conformation. limits its utility for our purpose. To determine the entire To locate a transition state, a stepwise procedure was reaction path of the substitution reaction, we calculated the followed. First, the gas-phase transition state was docked into structures of the following species: (1) the active site cavity the active site by superimposing the -CH2-COO- moiety of the modeled by the 14 residues and one water molecule; (2) the gas-phase transition state and the side chain carboxylate of active site containing 1,2-dichloroethane substrate (Fig. 2a); Asp-124. The transition state was optimized with the amino (3) the active site with the SN2 TS (Figs. 2b and 3); and (4) the acid side chains (including the crystallographic water) in a alkyl-enzyme ester product and leaving ClϪ that is held fixed state. Second, with the resulting transition state fixed, the between the NH groups of Trp-125 and Trp-175 (Fig. 2c). In amino acid side chains were optimized. Third, the transition all calculations, the Asp-124 is assumed to be ionized because state was optimized again with the optimized side chains fixed. the x-ray crystallographic studies indicate that Asp-124 is Lastly, both side chains and transition state were optimized ionized in the presence of the DCE substrate. together. Starting with the gas-phase transition state rather In the x-ray crystallographic structure of haloalkane deha- than the substrate ground state greatly simplifies the transi- logenase with bound 1,2-dichloroethane substrate (12–14), the tion-state search and shortens the computational time. The attacking oxygen of the side chain carboxylate of Asp-124 is same transition state is ensured when starting with substrate located approximately equidistant to the two carbon atoms of ground state. DCE (3.5 and 3.5 Å, as reported in the Protein Data Bank file). The PM3-calculated transition state inside the model cavity In the PM3-calculated structure of the active site containing (3; Figs. 2b and 3) is similar to the PM3-calculated gas-phase DCE substrate (2), the attacking carboxylate oxygen is 3.6 Å transition state for reaction of AcOϪ with DCE (16). The away from the DCE carbon undergoing reaction and 2.9 Å C ⅐⅐⅐O distance of the forming ester bond is 1.965 Å in 3, and away from the adjacent carbon as shown in Fig. 2a. The the corresponding C ⅐⅐⅐O distance in the transition state of difference between the calculated and the x-ray distances is the gas-phase reaction is 1.942 Å. Similarly, the C ⅐⅐⅐Cl probably due to the PM3 geometry corresponding to a single distance is 2.228 Å in 3, whereas the corresponding distance is 2.196 Å for the non-enzymatic reaction in the gas phase. Clearly, the transition state in the active site is slightly looser than the transition state for the non-enzymatic reaction in the gas phase. This is in agreement with previous theoretical observations that the transition state of SN2 reactions seems to be implastic (16, 22). In addition, the orientation of the remaining chlorine atom of DCE in the transition state is also similar. The Cl-C-C-Cl dihedral angle in the transition state of 3 is 67.4°, and it is 92.9° in the PM3-calculated gas-phase transition state of the non-enzymatic reaction. In the transition state of 3, the two NH ⅐⅐⅐Cl distances are 2.47 Å (to Trp-175) and 3.48 Å (to Trp-125), and the corresponding N ⅐⅐⅐Cl distances are 3.1 and 4.2 Å, respectively. To check whether the unequal N ⅐⅐⅐Cl distances were caused by the constraints imposed during the PM3 calculations, we reconstituted the calculated E⅐TS into the enzyme and carried out molecular mechanics optimization on the entire enzyme with bound crystallographic water molecules using QUANTA͞CHARMM (Micron Separations). During the molecular mechanical calculation, the forming O ⅐⅐⅐C and the breaking C ⅐⅐⅐Cl FIG. 1. The 14 amino acids that were included in the calculations. distances were kept fixed and no other constraints were used. The relative position of each residue is projected from the x-ray crystal However, after several hundred steps of energy minimization structure. we did not observe any indication that the two N ⅐⅐⅐Cl Downloaded by guest on September 28, 2021 Biochemistry: Lightstone et al. Proc. Natl. Acad. Sci. USA 94 (1997) 8419

FIG. 2. The three PM3-calculated stationary points for the chloride displacement from DCE by the carboxylate of Asp-124 in the active site of haloalkane dehalogenase. Only the amino acid residue side chains involved in the SN2 displacement and the substrate are shown. (A) The ES complex, where the nucleophilic oxygen is 3.57 Å from the electrophilic carbon. (B) The transition state. (C) The ester intermediate with the bound chloride between the indole HN of Trp-125 and Trp-175.

distances become equal. Further molecular dynamics simula- distances are 2.48 (to Trp-175) and 1.75 Å (to Trp-125), tions will be carried out to examine this aspect of the reaction. respectively. Again, molecular dynamics simulations with the Surprisingly, in the calculated ester product of the SN2 entire enzyme–product complex could provide more informa- reaction (4; Fig. 2c), the -OCH2CH2Cl group is in an almost tion on the hydrogen-bonding interactions between the chlo- eclipsed conformation as expressed by Cl-C-C-O dihedral ride ion and the two indole HN hydrogens. angle of Ϫ114.6°. This might be due to the fact that the model We also tried to estimate the potential energy barrier for the cavity has limited flexibility because of the fixed backbone SN2 reaction in the active site. However, it should be pointed atoms. The chloride anion product has moved between the two out that these energetic calculations may only be approxima- tryptophan indole NH hydrogens. However, the chloride ion is tions due to the restriction of the movement of the peptide much closer to the indole nitrogen atom of the Trp-125 backbone in the energy-minimization calculations. A recent residue. In the product, the Cl ⅐⅐⅐N distances are 3.54 Å (to study on carbonic anhydrase II indicated that the dynamic the indole nitrogen of Trp-175) and 2.82 Å (to the indole motion of the enzyme has a large effect on the calculated nitrogen of Trp-125) compared with the x-ray coordinate energetics (23). The calculated energies reveal a complexation distances of 3.2 and 3.5 Å, respectively. The Cl ⅐⅐⅐H-(N) energy of Ϫ15.4 kcal͞mol for the formation of the ES complex

FIG. 3. A stereoview of the PM3-calculated transition state active site with 14 amino acid residues surrounding the 1,2-dichloroethane substrate. This view of the active site is from within the enzyme, and the active site is completely sequestered from solvent. The partial bonds of the SN2 reaction are colored in magenta. Yellow-colored atoms of the peptide backbone are those fixed during the calculation. Those amino acid residue side chains that were allowed to move during the calculation are colored cyan. The amino acid residues involved in the reaction (Asp-124, Trp-125, and Trp-175) and the substrate are color-coded in the CPK standard: C, black; O, red; N, dark blue; H, white; and Cl, green. Downloaded by guest on September 28, 2021 8420 Biochemistry: Lightstone et al. Proc. Natl. Acad. Sci. USA 94 (1997)

(1 ϩ DCE 3 2). DCE is not well solvated in aqueous solution, sition-state structure of the enzyme haloalkane dehalogenase- and the experimental solvation free energy for DCE is only catalyzed reaction, it would be beneficial to investigate how the about Ϫ1.5 kcal͞mol (24). Therefore, it requires only approx- dynamic motion of the enzyme affects the calculated transi- imately 1.5 kcal͞mol free energy to bring DCE to the active site tion-state structure and the energetics. Further molecular of the enzyme. However, there is a large entropic penalty for dynamics and combined quantum mechanics and molecular the formation of the ES complex. The entropic cost of 3–6 mechanics calculations are underway to address this issue. kcal͞mol (T⌬S) to bring DCE to the active site can be estimated roughly from experimental entropies in solution for The assistance by Rhonda Torres in using QUANTA͞CHARMM is formation of complexes and in comparison of reactions of appreciated. We acknowledge the National Center for Supercomput- different kinetic order at 25°C (25). Thus, there is at least a ing Applications (Urbana, IL) for allocation of computing time. This 4.5–7.5 kcal͞mol free energy cost to bring DCE to the active work was supported by the National Institutes of Health. site. However, the binding energy gained is enough to compensate for this free energy cost. As a result, the free energy 1. Pauling, L. (1946) Chem. Eng. News 24, 1375–1377. of formation of the ES complex is probably less negative than 2. Jencks, W. P. (1966) in Current Aspects of Biochemical Energetics, ed. Kaplan, N. O. & Kennedy, E. P. (Academic, New York), pp. the above-mentioned value (Ϫ15.4 kcal͞mol) by at least 273–298. 4.5–7.5 kcal͞mol. The overall reaction is exothermic by 13.6 3. Wolfenden, R. (1969) Nature (London) 223, 704–705. kcal͞mol, and the overall potential energy barrier is about 17 4. Fersht, A. R. (1985) Enzyme Structure and Mechanism (Freeman, kcal͞mol. Remembering that the relationship of Asp-124– New York), 2nd Ed. Ϫ CO2 and DCE is that of a gas-phase ion–molecule complex, 5. Warshel, A. (1991) Computer Modeling of Chemical Reactions in it is interesting to note that the barrier (32.5 kcal ͞mol) from Enzymes and in Solution (Wiley, New York). the ES complex to the enzyme-bound TS ( 2 3 3) and the 6. Aqvist, J. & Warshel, A. (1993) Chem. Rev. 93, 2523–2544. barrier from the ion–molecule complex to the same transition 7. Daggett, V., Schroder, S. & Kollman, P. A. (1991) J. Am. Chem. state in the gas-phase nonenzymatic reaction (16) differ only Soc. 113, 8926–8935. J. Bacteriol. 163, by about 2 kcal mol. Our previous study indicated that the 8. Keuning, S., Janssen, D. B. & Witholt, B. (1985) ͞ 635–639. calculated gas-phase non-enzymatic reaction barrier using 9. Fetzner, S. & Lingens, F. (1994) Microbiol. Rev. 58, 641–685. PM3 is about 7.3 kcal͞mol higher than the corresponding 10. Janssen, D. B., van der Ploeg, J. R. & Pries, F. (1995) Environ. barrier calculated using ab initio molecular orbital theory at Health Perspect. 103, 29–32. the HF͞6–31ϩG(d) level (16). Thus, the PM3 barrier for the 11. Leisinger, T. (1996) Curr. Opin. Biotechnol. 7, 295–300. enzymatic reaction should be corrected by subtraction of Ϸ7.3 12. Verschueren, K. H. G., Seljee, F., Rozeboom, H. J., Kalk, K. H. kcal͞mol to provide Ϸ25 kcal͞mol. & Dijkstra, B. W. (1993) Nature (London) 363, 693–698. In the enzyme–substrate complex, the relative arrangement 13. Verschueren, K. H. G., Kingma, J., Rozeboom, H. J., Kalk, K. H., of DCE to the carboxylate of Asp-124 resembles the gas-phase Janssen, D. B. & Dijkstra, B. W. (1993) Biochemistry 32, 9031– ion–molecule complex of the non-enzymatic reaction. Weak 9037. 14. Verschueren, K. H. G., Franken, S. M., Rozeboom, H. J., Kalk, hydrogen bonding interactions between one of the DCE K. H. & Dijkstra, B. W. (1993) J. Mol. Biol. 232, 856–872. chlorines and the indole NHs of Trp-125 and Trp-175 along 15. Kennes, C., Pries, F., Krooshof, G. H., Bokma, E., Kingma, J. & with the ion–molecule complex electrostatic attraction hold Janssen, D. B. (1995) Eur. J. Biochem. 228, 403–407. the substrate in place. As the S N2 reaction proceeds, negative 16. Maulitz, A. H., Lightstone, F. C., Zheng, Y.-J. & Bruice, T. C. charge gradually accumulates on the leaving chlorine, (1997) Proc. Natl. Acad. Sci. USA 94, 6591–6595. strengthening hydrogen-bonding interactions between Cl␦Ϫ 17. Stewart, J. J. P. (1989) J. Comput. Chem. 10, 209–220. and the indole HNs. This differential hydrogen bonding could 18. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Gill, P. M. W., provide significant transition-state stabilization (12–14); sim- Johnson, B. G., et al. (1995) GAUSSIAN 94 (Gaussian, Pittsburgh), ilar effects have been proposed for other enzymatic reactions Revision B.2. (26–30). It has been shown recently that the non-enzymatic 19. Dewar, M. J. S., Zoebisch, E. G., Healy, E. F. & Stewart, J. J. P. (1985) J. Am. Chem. Soc. 107, 3902–3909. reaction is extremely slow in aqueous solution and the gas- 20. Zheng, Y.-J. & Merz, K. M., Jr. (1992) J. Comput. Chem. 13, phase reaction is very fast (16). Taken together, it is clear that 1151–1169. the haloalkane dehalogenase derives its catalytic power from: 21. Hartsough, D. & Merz, K. M., Jr. (1995) J. Phys. Chem. 99, (i) bringing the electrophile and nucleophile together in a 11266–11275. reactive orientation in a very low dielectric environment to 22. Shaik, S. S., Schlegel, H. B. & Wolfe, S. (1992) in Theoretical allow the reaction to occur without much structural reorgani- Aspects of Physical Organic Chemistry: The SN2 Mechanism zation (16), (ii) stabilizing the chloride anion-leaving group by (Wiley, New York), pp. 188–191. hydrogen bonding (with the indole NHs of Trp-125 and 23. Merz, K. M., Jr., & Banci, L. (1996) J. Phys. Chem. 100, 17414– Trp-175), and (iii) desolvation. Concerning our findings that 17420. the enzyme has been selected to provide the most favorable 24. Cabani, S., Gianni, P., Mollica, V. & Lepori, L. (1981) J. Solution Chem. 10, 563–598. environment for carboxylate nucleophilic attack and to stabi- 25. Bruice, T. C. (1970) The Enzymes (Academic, New York), Vol. lize the gas-phase transition state, we note that Dewar and 2, p. 217. Storch (31) have previously drawn attention to expected 26. Gerlt, J. A. & Gassman, P. G. (1993) Biochemistry 32, 11943– similarities between gas-phase and enzymatic SN2 reactions of 11952. negative nucleophile and neutral substrate. They pointed out 27. Zheng, Y.-J. & Ornstein, R. L. (1997) J. Am. Chem. Soc. 119, that such enzymatic reactions, like the gas-phase reactions, 648–655. should pass through reactive ion–molecule complex ground 28. Zheng, Y. -J. & Bruice, T. C. (1997) J. Am. Chem. Soc. 119, states and that the facility of the enzymatic reaction could be 3868–3877. Ϫ 29. Zheng, Y.-J. & Bruice, T. C. (1997) Proc. Natl. Acad. Sci. USA 94, explained by this feature. The SN2 displacement of Cl in the Xanthobacter autothropicus haloalkane dehalogenase enzyme 4285–4288. 30. Benning, M. M., Taylor, K. L., Liu, R.-Q., Yang, G., Xiang, H., appears to provide an example. It is well known that ion– Wesenberg, G., Dunaway-Mariano, D. & Holden, H. M. (1996) molecule SN2 displacements, as in Eq. 1, are much more rapid Biochemistry 35, 8103–8109. in the gas phase than in solution (32). 31. Dewar, M. J. S. & D. M. Storch, (1985) Proc. Natl. Acad. Sci. USA Although our model using 14 residues that form the active 82, 2225–2229. site cavity is a reasonable approximation to model the tran- 32. Nibbering, N. M. M. (1988) Adv. Phys. Org. Chem. 24, 1–55. Downloaded by guest on September 28, 2021