Research Kinetic Isotope Effects on Dehalogenations at an Aromatic Carbon AGNIESZKA DYBALA-DEFRATYKA,† LUKASZ SZATKOWSKI,† RAFAŁ KAMINSKI,† MONIKA WUJEC,‡ A G A T A S I W E K , ‡ A N D P I O T R P A N E T H * ,† Institute of Applied Radiation Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland, and Department of Organic Chemistry, Faculty of Pharmacy, Medical University, Staszica 6, 20-081 Lublin, Poland
Received January 28, 2008. Revised manuscript received May 26, 2008. Accepted June 1, 2008.
In order to interpret the observed isotopic fractionation it is necessary to understand its relationship with the isotope effect(s) on steps that occur during the conversion of the initial reactant to the final product. We examine this relationship from the biochemical point of view and elaborate on the consequences of the assumptions that it is based on. We illustrate the discrepancies between theoretical and experimental interpretation of kinetic isotope effects on examples of dehalogenation reactions that occur at an aromatic carbon atom. The examples include 4-chlorobenzoyl-CoA dehalogenasecatalyzed conversion of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA, dehaloperoxidase-catalyzed conversion of 2,4,6trichlorophenol to 2,6-dichloroquinone, and spontaneous hydrolysis of atrazine at pH 12. For this latter reaction we have measured the chlorine kinetic isotope effect and estimated its value theoretically at the DFT level of theory. Results of chlorine kinetic isotope effects suggest that the studied dechlorination reactions proceed in a single step with significant weakening of the carbon-chlorine bond in the transition state.
Introduction Isotopic fractionation can be used as a very informative tool in studies of mechanisms of chemical, biochemical, and physical processes that can be further used in basic sciences, as well as in applied areas such as drug design, environmental remediation, etc. However, its interpretation is frequently complicated by one or more factors. In order to draw conclusions from isotopic studies it is thus necessary to understand the relationship between the isotope effect on the step leading to the rate-determining transition state (the isotope effect associated with this step is called intrinsic) (1) and the observed overall isotopic fractionation. Recently, an excellent review on these problems has been published (2). Herein we examine this relationship from the biochemical point of view and elaborate on consequences of the assumptions that are made during its derivation. Information gained from intrinsic isotope effects is used in rational design * Corresponding author e-mail:
[email protected]. † Technical University of Lodz. ‡ Medical University. 7744
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 21, 2008
of inhibitors (3–6) that can be applied as drugs in medicine or pesticides in agriculture. In the second part, we discuss discrepancies between theoretical and experimental interpretation of kinetic isotope effects which sometimes lead to different conclusions (7). In particular, the example of the 4-benzoyl-CoA dehalogenasecatalyzed (BCD) reaction is presented. We show that different mechanistic scenarios for this reaction emerge from theoretical calculations and experimental measurements. A mechanism of BCD-catalyzed reaction is proposed based on the calculations of atrazine hydrolysis and the model reaction of dechlorination catalyzed by dehaloperoxidase (DHP). Atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6triazine) is an herbicide successfully applied in agriculture dating from the 1950s for fighting broadleaf and some grassy weeds (8). Biodegradation of atrazine is controlled by hydrolytic dehalogenation, although conjugation with peptides and N-dealkilations have been observed. Chlorinated aromatics are one of the widest group of compounds used in many industrial processes of high toxicity. They constitute a major environmental problem (9, 10). One example of such compounds are phenols and their polychlorinated derivatives coming from coal conversion, petrol purification, polymer, drug, dye, and pesticide production. However, their source is not only limited to anthropogenic activity. Less recognized, but common sources of haloaromatics are biotic (11–13). There are many groups of enzymes that have the ability to degrade halogenated compounds. Dehalogenating enzymes are the first line of defense against these toxicants.
Observed Vs Intrinsic Isotope Effects In order to consider all major contributions affecting the overall isotope effect that can be compared with the experimental value, the minimal mechanism necessary to be considered is presented as eq 1. It includes formation of the Michaelis-Menten complex (ES), chemical reaction step, leading to enzyme-bound products complex (EP), and subsequent release of the products. Since it is usually assumed that binding and transport processes do not introduce isotopic fractionation, the last step with rate constant k5 can be considered as a combination of steps relating to the release of the product from the enzyme and its transport in the environment to the site where it is recovered. k1
k3
k2
k4
k5
E + S a ES a EP a K + P
(1)
Assumption of steady-state concentrations of complexes ES and EP, and the above-mentioned assumption that only one “chemical step” (e.g., the chemical conversion in the active site of the enzyme) is isotope-sensitive, leads to the expression for the overall isotope effect:
( ) ( )
k3 KIE3 k5 + KIE3 + KIE3 + cf + EIE3⁄4cr KIE4 k4 k2 ) (2) KIEapp ) k5 k3 1 + cf + cr 1+ 1+ k4 k2 In this equation, KIE3/KIE4 corresponds to the equilibrium isotope effect, EIE3/4 corresponds to the step characterized by the rate constants k3 in the forward and k4 in the reverse direction. Also cf ) k3/k2 is defined as the commitment for catalysis for the reaction that proceeds from the substrate to product, while the ratio k4/k5 is the commitment for catalysis for the reaction that proceeds from products to substrates, 10.1021/es800276y CCC: $40.75
2008 American Chemical Society
Published on Web 07/04/2008
and is therefore called reverse commitment to catalysis and marked as cr. Note that since we have assumed that only these steps are isotopically sensitive, EIE3/4 corresponds to the overall equilibrium isotope effect. When the chemistry step is irreversible then eq 2 can be further simplified to KIEapp )
KIE3 + cf 1 + cf
(3)
Interpretation of the experimental data based on eqs 2 or 3 is uncertain since we usually do not know exact intrinsic value(s) and the partitioning factor(s). In addition, both experimental and theoretical approaches carry uncertainties that are frequently unrecognized or ignored. Below we consider the most common factors that cause this uncertainty. Weaknesses of the Experimental Data. In obtaining eq 2, we have assumed that there is only one substrate and only one isotope-sensitive step. This obviously is seldom true. For example, it is known that binding processes may exhibit isotope effects (14), although they are usually much smaller than primary KIEs and thus may be neglected in the first approximation. Similarly, small isotopic fractionation may be associated with physical processes such as transport (as documented for example by the isotopic fractionation associated with chromatography) (15–17). Second, we have neglected nonenzymatic reaction proceeding in parallel to the enzymatic process. Again, usually such chemical reaction (e.g., hydrolysis) proceeds much slower than the corresponding enzymatic one and can be ignored. If, however, it cannot be neglected the expressions for eqs 2 and 3 become much more complicated (see, for example, the case of carbon KIEs for carbonic anhydrase) (18). Furthermore, irreversibility of the isotope-sensitive step is frequently not true and commitments for the reverse direction of the reversible reactions have to be included. All these corrections can be added quite easily, and the corresponding equation can be dealt with analytically if the actual scheme can be derived. However, unequivocal solution might require measurements of a number of different isotope effects (of different atoms or of the same atom under different conditions, such as temperature, pH, concentrations, etc.) (18, 19). Compound Specific Isotopic Analysis (CSIA). An important issue in environmental studies is the source of the isotopic information. Very often in such studies measured isotopic composition is associated with a compound rather than with a particular atom within a molecule. This so-called compound specific isotopic analysis (CSIA) averages isotopic composition of all atoms of the particular element regardless of their chemical positions. If only one of these positions is associated with a sizable (primary) KIE, then isotopic fractionation is diluted and this should again be taken into account. However, the assumption, that other positions yield no isotopic fractionation and can be considered as diluting the primary KIE only, is usually not true. This is very nicely illustrated by the deuterium isotope effect on phenylalanine ammonia lyase reaction. One of the suggested mechanisms assumes dearomatization of the phenyl ring due to formation of the covalent bond at the ortho position with a prostetic group of the enzyme (20). Since in the other mechanism (21) the benzene ring remains intact, secondary deuterium KIE seemed to be a good tool for distinguishing these mechanisms. For the first mechanisms an inverse KIE of the hydrogen in ortho position was expected, whereas no or a very small normal KIE was expected for the other mechanism. Experiments with phenyl-d5-substituted reactant yielded substantially normal KIE (larger than unity). However, the experiment with tritium in ortho positions only yielded an inverse KIE (less than unity). Theoretical evaluations of KIEs of all five hydrogen atoms of the phenyl ring revealed (22) that, in fact, KIE of hydrogen bonded to the carbon atom
FIGURE 1. Schuster and Retey’s mechanism of action of phenylalanine ammonia-lyase. that undergoes sp2 to sp3 transformation is inverse. However, cumulative isotope effect of the other four hydrogen atoms renders the overall isotope effect observed for the phenyl-d5 derivative positive; position specific deuterium KIEs for the first mechanism are marked in Figure 1. Finally, an important approximation that has been invoked in derivation of eqs 2 and 3 is the steady-state approximation. It allows for mathematical simplification of the kinetic equations which otherwise become much more complicated and frequently only numerically solvable. When this approximation is not valid, the observed isotope effect changes as the reaction progresses (23, 24). A similar situation is also observed when a side reaction is present (25, 26). Weaknesses of the Theoretical Data. Intrinsic KIEs are usually calculated from the Bigeleisen equation (27): * * · sinh(uiL ⁄ 2) kL νL* 3n-6 uiL · sinh(uiH ⁄ 2) 3n -7 uiH ) *· · (4) * * kH ν u · sinh(u ⁄ 2) u · sinh(u ⁄ 2) iH iL i i H
∏
*
∏
iL
iH
where u ) hν/kBT, h and kB are Planck and Boltzmann constants, respectively, and T is the absolute temperature. Also, n is the number of atoms, and vi are the frequencies of normal modes of vibrations. The superscript “*” indicates the properties of the transition state. However, this equation is derived within the transition state theory framework. This theory does not include some nonclassical effects. The one that has the strongest bearing on the isotope effects is tunneling. Even in enzymatic reactions this phenomenon may change dramatically the value of isotope effects, especially in the case of hydrogen isotope effects. Variational transition state theory should be employed in such cases (28). Furthermore, the choice of theory level and basis set used in these calculations will influence the value of the isotope effect (29–31). Another problem is the use of harmonic approximation. Fortunately anharmonicity tends to diminish the value of an isotope effect, while inclusion of tunneling increases it. Neglecting both of these, some extent of canceling exists for heavy atom isotope effects. Another important source of error is treatment of the reaction environment. When a substrate in solution is considered, the solvent is frequently modeled by the implicit solvent model, in which it is represented by a continuum dielectric medium. Since no explicit solvent molecules (opposite to the explicit solvent models) are used, the calculations are not too demanding in terms of computational resources. It should be kept in mind, however, that this approach can lead to completely wrong isotopic results if interactions that are neglected in these models are crucial to isotopic fractionation. An enzymatic environment, on the other hand, is usually included in calculations at a theory level lower than the one used for the enzyme active site. This formalism is referred to as QM/MM calculations because usually a quantum theory level is used in the description of the reaction and active site residues, whereas molecular mechanics is used in the enzyme description. This very economical approach has, however, its weak points. For example, it is not always straightforward VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7745
FIGURE 2. Alternative pathways of 4-chlorobenzoyl-CoA dehalogenase-catalyzed reaction.
FIGURE 3. Mechanism of atrazine hydrolysis. to decide where the border should be placed between the parts of the system described by different theory levels and how the interactions between them should be treated. Unfortunately, as pointed out recently by Morokuma and co-workers (32), these problems may significantly influence the results.
Experimental section Measurements of Chlorine KIE. Nitric acid (pure p.a.), silver nitrite (pure p.a.), sodium bicarbonate (pure p.a.), citric acid (pure p.a.), potassium nitrite (pure p.a.), ethanol (special pure), chloroform (pure p.a.), and sodium phosphate (pure p.a.) from POCh Gliwice, mercuric (II) thiocyanate (pure p.a., Fluka), ferric ammonium sulfate (L.P.P-H ”OCh” Lublin, Poland), concentrated sulfuric acid (suprapur, Merck) calcium oxide (pure p.a., BDH Chemicals Ltd.), and 4-methyl2-pentanone (spectrophotometric grade, Sigma-Aldrich) were used without further purification. Atrazine (pure p.a., ZChO-Azot, Jaworzno) was purified by extracting into trichloromethane, washed four times with water, dried with magnesium sulfate, precipitated with n-hexane, and dried in the desiccator (33). The resulting crystals were shown chromatographically (TLC, DC-Alufolien Kieselgel 60 Merck, chloroform: acetone 4:1) to be pure with melting point of 175-176 °C. The hydrolysis of atrazine (8 mM) was carried out in the saturated solution of calcium hydroxide at pH 12 and temperature of 21 °C in the dark. Because of weak solubility of atrazine in pure water a small amount of ethanol (0.6% v:v) was added. The progress of the reaction was monitored by measuring the concentration of chloride ions using the colorimetric method (34). At the desired reaction progress (10-25% of conversion), the reaction was stopped by addition of concentrated nitric acid to reach pH of about 7.5, the 7746
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 21, 2008
solution was then concentrated to the volume of about 50 mL, and washed twice with 10 mL of 4-methyl-2-pentanone in order to remove the remaining atrazine. Then the solution was acidified and silver chloride was precipitated with AgNO3 at about 80 °C. After 24 hours, the precipitate was drained and washed with nitric acid. The washing procedure was repeated three times and then AgCl was left to dry in a desiccator in the dark. Chlorine kinetic isotope effect was calculated from the following equation (27): k35 ln(1 - f ) ) k37 Rf ln 1 - f R0
(
)
(5)
where f is the reaction progress, Rf is the chlorine isotopic ratio of the product at the reaction progress f, and R0 is the chlorine isotopic ratio of the initial atrazine. Chlorine isotopic ratios were measured using hybrid FAB-IR mass spectrometer as described earlier (35) with modified sample support that results in reduced sample requirement (to be published elsewhere). R0 is determined using AgCl precipitated in the same way as described above from chloride obtained from atrazine by the Lassaigne method (36). Computational Part. All calculations were carried out using Gaussian 03 (37) at the DFT level using BP86 functional (38, 39). The basis set used was 6-31G (40–42) augmented with a single set of diffuse functions (43), a set of d functions on heavy atoms, and a set of p functions on hydrogen atoms (44)(6-31+G**) due to the presence of the ionic product. The influence of the environment was modeled using the polarizable continuum solvent model (PCM) (45) with parameters for water in case of aqueous solutions and with parameters for ether (dielectric constant ) 4.3) for DHP to account for low polarity of its active site. Molecular geometries were fully optimized and vibrational analysis was carried out to confirm identity of the stationary points (3n - 6 real vibrations in the case of reactants and one imaginary frequency corresponding to the desired reaction coordinate in the case of transition state structures). Calculations of KIEs were performed using our ISOEFF98 program (46). KIEs were obtained from the complete Bigeleisen equation (27) at 300 K for the transition from
FIGURE 4. Transition state structure of the atrazine hydrolysis reaction. Left: front view with atom numbering. Right: view with the triazine ring perpendicular to the plane. proximity complexes of both reactants to the corresponding transition state.
Computational Results for Atrazine and Trichlorophenol As mentioned in the introduction, the major goal in applying isotopic fractionation to chemical and biochemical processes is elucidation of the transition state structure of the ratelimiting step from the intrinsic isotope effect. The information can also be obtained from molecular modeling. Sometimes, however, interpretation of the results from these two sources leads to mutually excluding conclusions. We have analyzed such cases for a simple SN2 reaction, in which C-Cl bond is being broken (7, 47, 48). Similar discrepancies have been observed in the case of enzymatic dehalogenation at the aromatic carbonsthe process quite frequently observed in many environmentally important reactions. In our studies of the 4-chlorobenzoyl-CoA dehalogenase-catalyzed (CBD) reaction (49), we have measured chlorine kinetic isotope effect of 1.0090. This large value of the chlorine KIE indicates that the carbon-chlorine bond breaking occurs in the ratedetermining step. Within the accepted mechanism of the 4-chlorobenzoyl-CoA dehalogenase reaction, presented in the upper part of Figure 2, the decomposition of the Meisenheimer complex (E · MC) should dominate the ratedetermining steps. However, recent calculations (50) indicated that it is formation of this complex that is rate-limiting. Calculations using optimized geometries of the E · S complex and the transition state for the E · MC formation yielded chlorine KIE of only 1.0004 (unpublished results) in strong disagreement with the experimental value. Thus far, this discrepancy has not been resolved. Our calculations on a model of the reaction catalyzed by dehaloperoxidase and on the hydrolysis of atrazine that are presented below lead to new mechanistic suggestions that may accommodate results from both computational modeling and experimental determination of the chlorine KIEs. Dehalogenation is the rate-limiting step of atrazine biodegradation (51) in bacteria, e.g., Pseudomonas sp. In higher plants, such as corn, the dehalogenation process is catalyzed by benzoxazinone (2,4-dihydroxy-3-keto-7-methoxy-1,4-benzoxazine) (52). Atrazine hydrolysis has been studied recently at various pH conditions (53). Given the wide interest in the mechanism of its hydrolysis and the fact that this process involves either an intermediate or the transition state, where the structure has to be similar to the
Meisenheimer complex, we have studied this reaction theoretically and experimentally. Our calculations, carried out at the BP86/6-31+G** level of theory, indicate that this reaction proceeds in a single step as illustrated in Figure 3. The energetic barrier of this process in aqueous solution was found to be equal to 31.8 kcal/mol in good agreement with the experimental data (54). The transition state structure is presented in Figure 4 together with atom numbering. The key geometrical features of this transition state are available in Table S1 of the Supporting Information and compared to the substrate. Geometrical changes are constrained to the small fragment of the triazine ring. The major change is observed in C-Cl bond distancesit increases by about 0.5 Å. The forming CsO bond is also very long, about 0.3 Å longer than in the product. Changes in other bond distances, on the other hand, are negligible. The preliminary value of the chlorine kinetic isotope effect for this reaction at pH 12 is equal to 1.0069 ( 0.0005 (SE reported). This quite typical value (55) indicates substantial CsCl bond loosening in the transition state and is in reasonable agreement with the value predicted theoretically (see Table S1 in the Supporting Information). Isotope effects of atoms in water molecule are also of interest. The hydrogen KIE of the atom that does not interact with the departing chloride exhibits significantly inverse isotope effect, whereas the one that interacts with the chloride shows very large normal isotope effect of over 20% of the oxygen atom of water molecule. Unfortunately, these two protons are easily exchangeable and thus their application in mechanistic studies is impractical. Oxygen on the other hand is a promising site; one would expect an inverse isotope effect if only a new CsO bond is formed. The slightly positive value reported in Table 1 reflects advanced breaking of one of the OsH bonds in the transition state. The presented calculations provide an opportunity to illustrate how CSIA influences the observed isotopic fractionation; we have calculated isotope effects of every atom of the reacting system. These individual, position specific, values are collected in Table 1. The last row (KIECSIA) shows values that correspond to the isotopic fractionation that would result from the CSIA analysis. It is worth noticing that the carbon KIE of about 3% (of the C6 atom) is significantly diluted by isotopic fractionation of other positions. The resulting isotopic fractionation that would be detected with CSIA is about 1.0064. The carbon KIE data also illustrate that VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7747
TABLE 1. Position Specific Isotope Effects of All Atoms of the Transition State. Atom Numbering Taken from Figure 4 N 2 4 5 7 9
1.00374 0.99979 1.00226 1.00047 0.99985
KIECSIA a
C
1.0012
1 3 6 11 14 18 20 24
Cl 1.00186 1.00182 1.02859 0.99998 0.99990 0.99995 0.99990 0.99992
1.0064
28
Haqa
H 1.00819
1.0082
8 10 12 13 15 16 17 19 21 22 23 25 26 27
0.99934 0.99870 0.99856 0.99326 1.00044 0.99525 0.99589 1.00572 0.99989 0.99664 0.99983 0.99983 0.99735 0.99987 0.9984
30 31
O 0.89399 1.23427
1.0502
29
1.00344
1.0034
Hydrogen atoms of water molecule.
FIGURE 5. Mechanism of trichlorophenol hydrolysis catalyzed by DHP. the assumption that only one position shows isotopic fractionation is an oversimplification. In the studied case, both the C1 and C3 positions also contribute to observed fractionation. Similar mechanistic conclusions emerge from our theoretical studies of the enzymatic dechlorination at the aromatic carbon in dehaloperoxidase-catalyzed (DHP) conversion of 2,4,6-trichlorophenol to 2,6-dichloroquinone. DHP is an unusual heme-containing protein with a globin fold and peroxidase activity. It is capable of the binding of molecules such as oxygen and carbon monoxide, and has catalytic properties similar to those of horse radish peroxidase (HRP) (56). DHP performs two-electron oxidations in the active site, and depending on the pH of the reaction, it can adopt a different mechanism, which is quite a striking feature in the peroxidase family of enzymes. Kinetic studies using different protonation states of substrate have been performed, however the mechanistic details remain unknown (57), especially the effect of the pH on the dehalogenation step. The dehalogenation occurs in the active site as the result of reaction between water molecule and the radical cation of the substrate as schematically presented in Figure 5. We have modeled the dehalogenation step using two different forms of substrate: deprotonated and protonated radical cations, and we have obtained the transition state structures (Figure 6) in which neutral water molecule attacks the C4-carbon atom, which is similar to the results obtained for the atrazine hydrolysis. Chlorine and carbon isotope effects obtained for this reaction are similar to those obtained for the atrazine hydrolysis; 1.0086/1.0073 and 1.0346/1.0375 for deprotonated/protonated forms, respectively. Unlike the atrazine hydrolysis, however, the oxygen KIE on the DHPcatalyzed dehalogenation is inverse (0.9932/0.9727). As mentioned above, the inverse value indicates that O-H bonds remain mostly intact on the way from the reactant to the transition state. Thus 18O-KIE seems to be a good measure of the amount of deprotonation of the water acting as the nucleophile in the transition state. This is promising since recently measurements of the incoming group oxygen isotope effects became available (58). 7748
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 21, 2008
FIGURE 6. Transition state structure of the model of the DHP-catalyzed reaction. Modeling and isotope effects discussed above open a new avenue to the interpretation of the chlorine KIEs on the CBDcatalyzed reaction; the possibility of a tight SN2Ar transition state should be considered as the mechanistic alternative to the Meisenheimer complex formation. Since this isotope effect is amenable for determination further experimental and theoretical studies are under way to test this hypothesis.
Acknowledgments Studies of environmentally pertinent enzymatic dehalogenations are being supported over several years by a series of grants, most recent being 3 T09B 007 29 (ADD) and PBZMEiN-3/2/2006 (PP). Access to supercomputer facilities at PCCP (Poznan), ICM (Warsaw) and Cyfronet (Cracow) is acknowledged.
Supporting Information Available Key geometric features of atrazine and the transition state of its hydrolysis. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Dybala-Defratyka, A.; Kwiecien, R. A.; Sicinska D.; Paneth, P. Intrinsic Isotope Effectssthe Holy Graal of Studies of EnzymeCatalyzed Reactions. In Modelling Molecular Structure and Reactivity in Biological Systems; Brady, J., Gao, J., Naidoo, K. J., Hann, M., Field, M. , Eds.; Royal Society of Chemistry (UK) Special Publication S.: 2006, no. 303, 163-173.
(2) Elsner, M.; Zwank, L.; Hunkeler, D.; Schwarzenbach, R. P. A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environ. Sci. Technol. 2005, 39, 6896–6916. (3) Singh, V.; Evans, G. B.; Lenz, D. H.; Mason, J. M.; Clinch, K.; Mee, S.; Painter, F. G.; Tyler, P. C.; Furneaux, R. H.; Lee, J. E.; et al. Femtomolar transition state analogue inhibitors of 5′methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli. J. Biol. Chem. 2005, 280, 18265–18273. (4) Kwiecien, R. A.; Lewandowicz, A.; Paneth, P. Substrate-Enzyme Interactions from Modeling and Isotope Effects. In Challenges and Advances in Computational Chemistry and Physics; Leszczyn ˜ ski, J. In Molecular Materials with Specific Interactins; Sokalski, W. A.; 2007; Vol 4, pp 341-364. (5) Lee, J. E.; Singh, V.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Cornell, K. A.; Riscoe, M. K.; Schramm, V. L.; Howell, P. L. Structural rationale for the affinity of pico- and femtomolar transition state analogues of Escherichia coli 5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase. J. Biol. Chem. 2005, 280, 18274–18282. (6) Lewandowicz, A.; Shi, W.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Basso, L. A.; Santos, D. S.; Almo, S. C.; Schramm, V. L. Over-the-barrier transition state analogues and crystal structure with Mycobacterium tuberculosis purine nucleoside phosphorylase. Biochemistry 2003, 42, 6057–6066. (7) Fang, Y. r.; Gao, Y.; Ryberg, P.; Eriksson, J.; Kolodziejska-Huben, M.; Dybala-Defratyka, A.; Danielsson, R.; Paneth, P.; Matsson, O.; Westaway, K. C. Experimental and theoretical multiple kinetic isotope effects for an SN2 reaction. An attempt to determine transition-state structure and the ability of theoretical methods to predict experimental kinetic isotope effects. Chem. Eur. J. 2003, 9, 2696–2709. (8) Gysin, H.; Knuesli, E. Chemistry and Herbicidal Properties of Triazine Derivatives. In Advances in Pest Control Research; Metcalf, R. , Ed.; Wiley: New York, 1960; Vol. 3, 289-358. (9) Robinson, G. K.; Lenn, M. J. The bioremediation of polychlorinated biphenyls (PCBs): Problems and perspectives. Biotechnol. Genet. Eng. Rev. 1994, 12, 139–188. (10) Fetzner, S.; Lingens, F. Bacterial dehalogenases: Biochemistry, genetics, and biotechnological applications. Microbiol. Rev. 1994, 58, 641–685. (11) Faulkner, D. J. Marine natural products: metabolites of marine invertebrates and biotechnological applications. Nat. Prod. Rep. 1984, 1, 551–598. (12) Fenical, W. Halogenation in the Rhodophyta. Review. J. Phycol. 1975, 11, 245–259. (13) Gribble, G. W. The natural production of chlorinated compounds. Environ. Sci. Technol. 1994, 28, 310A–319A. (14) Rakowski, K.; Paneth, P. Isotope effects on binding. J. Mol. Struct. 1996, 378, 35–43. (15) Caimi, R. J.; Brenna, J. T. Quantitative evaluation of carbon isotopic fractionation during reversed-phase high-performance liquid chromatography. J.Chromatogr., A 1997, 757, 307–310. (16) Zhang, Y.-H.; Gunji, S.; Nomura, M.; Fujii, Y.; Oi, T. Observation of cerium isotope fractionation in ion-exchange chromatography of Ce(III)-malate complex. J. Chromatogr., A 2005, 1069, 133–139. (17) Smittenberg, R. H.; Sachs, J. P. Purification of dinosterol for hydrogen isotopic analysis using high-performance liquid chromatography-mass spectrometry. J. Chromatogr., A 2007, 1169, 70–76. (18) Smiley, J. A.; Paneth, P.; O’Leary, M. H.; Bell, J. B.; Jones, M. E. Investigation of the enzymic mechanism of yeast orotidine5′-monophosphate decarboxylase using carbon-13 kinetic isotope effects. Biochemistry 1991, 30, 6216–6223. (19) Sicinska, D.; Truhlar, D. G.; Paneth, P. Dependence of transition state structure on substrate: the intrinsic C-13 kinetic isotope effect is different for physiological and slow substrates of the ornithine decarboxylase reaction because of different hydrogen bonding structures. J. Am. Chem. Soc. 2005, 127, 5414–5422. (20) Schuster, B.; Retey, J. The mechanism of action of phenylalanine ammonia-lyase: The role of prosthetic dehydrolanine. Proc. Natl. Acad. Sci. 1995, 92, 8433–8437. (21) Hanson, K. R.; Havir, E. A. L-phenylalanine ammonia lyase. IV. Arch. Biochem. Biophys. 1970, 141, 1–17. (22) Lewandowicz, A.; Jemielity, J.; Kanska, M.; Zon, J.; Paneth, P. Tritium secondary kinetic isotope effect on phenylalanine ammonia-lyase-catalyzed reaction. Arch. Biochem. Biophys. 1999, 370, 216–221. (23) Paneth, P. On the application of the steady state to kinetic isotope effects. J. Am. Chem. Soc. 1985, 107, 7070–7071.
(24) Kaminski, Z.; Paneth, P.; Rudzinski, J. A. Study on the activation of carboxylic acids by means of 2-chloro-4,6-dimethoxy-1,3,5triazine and 2-chloro-4,6-diphenoxy-1,3,5-triazine. J. Org. Chem. 1998, 63, 4248–4255. (25) Gawlita-Kamzurska, E.; Paneth, P. Time-dependence of kinetic isotope effects of parallel reactions. Pol. J. Chem. 1992, 66, 209– 213. (26) Van Breukelen, B. M. Extending the Rayleigh equation to allow competing isotope fractionating pathways to improve quantification of biodegradation. Environ. Sci. Technol. 2007, 41, 4004–4010. (27) Melander, L.; Saunders, W. H., Jr. Reaction Rates of Isotopic Molecules; Wiley and Sons: NewYork, 1980. (28) Dybala-Defratyka, A.; Paneth, P.; Truhlar, D. G. Quantum Catalysis in Enzymes. In Quantum Tunneling in Enzyme Catalyzed Reactions; Scrutton, N., Allemann, R., Eds.; RSC Publishing: Cambridge, UK, 2008, in press. (29) Glad, S. S.; Jensen, F. Basis set and correlation effects on transition state geometries and kinetic isotope effects. J. Phys. Chem. 1996, 100, 16892–16898. (30) Matsson, O.; Dybala-Defratyka, A.; Rostkowski, M.; Paneth, P.; Westaway, K. C. A Theoretical investigation of a´-carbon kinetic isotope effects and their relationship to the transition-state structure of SN2 reactions. J. Org. Chem. 2005, 70, 4022–4027. (31) Dybala-Defratyka, A.; Rostkowski, M.; Matsson, O.; Westaway, K. C.; Paneth, P. A New Interpretation of chlorine leaving group kinetic isotope effects; A theoretical approach. J. Org. Chem. 2004, 69, 4900–4905. (32) Zheng, G.; Witek, H. A.; Bobadova.Parvanova, P.; Irle, S.; Musaev, D. G.; Prabhakar, R.; Morokuma, K.; Lundberg, M.; Elstner, M.; Ko¨hler; et al. Parameter calibration of transition-metal elements for the spin-polarized self-consistent-charge density-functional tight-binding (DFTB) method: Sc, Ti, Fe, Co, and Ni. J. Chem. Theory Comput. 2007, 3, 1349–367. (33) Wang, Y.; Duh, J.; Lin, K.; Chen, Y. Movement of three s-triazine herbicides atrazine, simazine, and ametryn in subtropical soils. Bull. Environ. Contam. Toxicol. 1996, 57, 743–750. (34) Iwasaki, I.; Utsumi, S.; Hagino, K.; Ozawa, T. A new spectrophotometric method for the determination of small amounts of chloride using the mercuric thiocyanate method. Bull. Chem. Soc. Jpn. 1956, 29, 860–864. (35) Westaway, K. C.; Koerner, T.; Fang, Y.-R.; Rudzinski, J.; Paneth, P. A new method of determining chlorine kinetic isotope effects. Anal. Chem. 1998, 70, 3548–3552. (36) Widmark, G. Systematic microdetection of arsenic, bromine, carbon, chlorine, fluorine, iodine, nitrogen, phosphorus, and sulfur in organic compounds by a modified Lassaigne’s method. Acta Chem. Scand. 1953, 7, 1395–1406. (37) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai,H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (38) Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A 1988, 38, 3098– 3100. (39) Perdew, J. P. Density-functional approximation for the correlation energy of the inhomogeneous electron gas. Phys. Rev. B 1986, 33, 8822–8824. (40) Hariharan, P. C.; Pople, J. A. The influence of polarization functions on molecular orbital hydrogenation energies. Theor. Chim. Acta 1973, 28, 213–222. (41) Ditchfield, R.; Hehre, W. J.; Pople, J. A. Self-consistent molecularorbital methods. IX. An extended Gaussian-type basis for molecular-orbital studies of organic molecules. J. Chem. Phys. 1971, 54, 724–728. VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7749
(42) Francl, M. M.; Pietro, W. J.; Hehre, W. J.; Binkley, J. S.; Gordon, M. S.; DeFrees, D. J.; Pople, J. A. Self-consistent molecular orbital methods. XXIII. A polarization-type basis set for second-row elements. J. Chem. Phys. 1982, 77, 3654–3665. (43) Clark, T.; Chandrasekhar, J.; Spitznagel, G. W.; Schleyer, P. v R. Efficient diffuse function-augmented basis sets for anion calculations. III. The 3-21+G basis set for first-row elements, Li-F. J. Comput. Chem. 1983, 4, 294–301. (44) Frisch, M. J.; Pople, J. A.; Binkley, J. S. Self-consistent molecular orbital methods 25. Supplementary functions for Gaussian basis sets. J. Chem. Phys. 1984, 80, 3265–3269. (45) Miertus, S.; Scrocco, E.; Tomasi, J. Electrostatic interaction of a solute with a continuum. A direct utilizaion of ab initio molecular potentials for the prevision of solvent effects. Chem. Phys. 1981, 55, 117–129. (46) Anisimov, V.; Paneth, P. ISOEFF98. A program for studies of isotope effects using Hessian modifications. J. Mathem. Chem. 1999, 26, 75–86. (47) Matsson, O.; Persson, J.; Axelsson, B. S.; Langstrom, B.; Fang, Y.-r.; Westaway, K. C. Using incoming group 11C/14C kinetic isotope effects to model the transition states for the SN2 reactions between para-substituted benzyl chlorides and labeled cyanide ion. J. Am. Chem. Soc. 1996, 118, 6350–6354. (48) Westaway, K. C.; Fang, Y.-r.; Person, J.; Matsson, O. Using 11C/ 14C incoming group and secondary a ´ -deuterium KIEs to determine how a change in leaving group alters the structure of the transition state of the SN2 reactions between mchlorobenzyl para-substituted benzenesulfonates and cyanide ion. J. Am. Chem. Soc. 1998, 120, 3340–3344. (49) Lewandowicz, A.; Rudzinski, J.; Luo, L.; Dunaway-Mariano, D.; Paneth, P. Determination of the chlorine kinetic isotope effect on the 4-chlorobenzoyl-CoA dehalogenase-catalyzed ncleophilic aromatic substitution. Arch. Biochem. Biophys. 2002, 398, 249– 252.
7750
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 21, 2008
(50) Xu, D. G.; Wei, Y. S.; Wu, J. B.; Dunaway.Mariano, D.; Guo, H.; Cui, Q.; Gao, J. L. QM/MM studies of the enzyme-catalyzed dechlorination of 4-chlorobenzoyl-CoA provide insight into reaction energetics. J. Am. Chem. Soc. 2004, 126, 13649–13658. (51) Wackett, L. P.; Sadowsky, M. J.; Martinez, B.; Shapir, N. Biodegradation of atrazine and related s-triazine compounds: from enzymes to field studies. Appl. Microbiol. Biotechnol. 2002, 58, 39–45. (52) Shimabukuro, R. H.; Swanson, H. R.; Walsh, W. C. Glutathione conjugation. Atrazine detoxication mechanism in corn. Plant Physiol. 1970, 46, 103–107. (53) Meyer, A.; Penning, H.; Elsner, M. Accurate compound specific carbon and nitrogen isotope analysis of atrazine: critical role of combustion oven conditions Environ. Sci. Technol. 2008, 42, 7757-7763. (54) Plust, S. J.; Loehe, J. R.; Feher, F. J.; Benedict, J. H.; Herbrandson, H. F. Kinetics and mechanism of hydrolysis of chloro-l,3,5triazines. Atrazine. J. Org. Chem. 1981, 46, 3661–3665. (55) Paneth, P. Chlorine Isotope Effects in Biological Systems. In Isotope Effects in Chemistry and Biology, Chapter 35; Kohen, A., Limbach, H. H. , Eds.; CRC Press, Inc: Baton Rouge, 2006; 875891. (56) Ferrari, R. P.; Laurenti, E.; Trotta, F. Oxidative 4-dechlorination of 2,4,6-trichlorophenol catalyzed by horseradish peroxidase. J. Biol. Inorg. Chem. 1999, 4, 232–237. (57) Franzen, S.; Gilvey, L. B.; Belyea, J. L. The pH dependence of the activity of dehaloperoxidase from Amphitrite ornate. Biochim. Biophys. Acta 2007, 1774, 121–130. (58) Cassano, A. G.; Anderson, V. E.; Harris, M. E. Evidence for direct attack by hydroxide in phosphodiester hydrolysis. J. Am. Chem. Soc. 2002, 124, 10964–10965.
ES800276Y
