Chem. Res. Toxicol. 1996, 8, 481-488
481
Articles Quantitative Structure-Activity Relationships Based on Computer Calculated Parameters for the Overall Rate of Glutathione S-Transferase Catalyzed Conjugation of a Series of Fluoronitrobenzenes Ivonne M. C. M. Rietjens," Ans E. M. F. Soffers, Guido J. E. J. Hooiveld, Cees Veeger, and Jacques Vervoort Department of Biochemistry, Agricultural University, DreGenlaan 3, 6703 HA Wageningen, The Netherlands Received November 18, 1994@
The present study describes quantitative structure-activity relationships (QSAR's) for the overall rate of conjugation of a series of fluoronitrobenzenes catalyzed by cytosolic glutathione S-transferases based on experimental data and outcomes of computer calculations. The natural logarithm of the rate of conjugation of the series of fluoronitrobenzenes correlates ( r = -0.986) with the calculated energy ( E )of their lowest unoccupied molecular orbital (LUMO) and also ( r = -0.987) with the relative heat of formation (AAHF) for formation of the Meisenheimer complex of the fluoronitrobenzenes with a MeS- model nucleophile. In addition, the paper describes QSAR's for the chemical reaction of glutathione with the fluorinated nitrobenzenes both at pH 7.6 and a t pH 9.9. These QSAR's are parallel to the one obtained for the enzyme catalyzed conversions. This indicates that in the overall reaction (both chemical and enzyme catalyzed) the interaction between the thiolate anion of glutathione and the fluoronitrobenzene leading to the Meisenheimer reaction intermediate is the rate-limiting step in overall conversion of these substrates. The parallel QSAR's of the chemical and enzymatic reaction also indicate that in the enzymatic reaction chemical reactivity parameters determine the overall outcome of catalysis and, in addition, that the chemical and enzymatic reactions proceed through a similar reaction pathway with comparable reaction intermediates. Additional results of the present study demonstrate that the regioselectivity of the glutathione conjugation cannot be explained on the basis of calculated characteristics of the LUMO of the fluoronitrobenzenes or the AAHF for the formation of their Meisenheimer reaction complex. Taken together, the results of the present study demonstrate that the number of fluorine substituents in the fluoronitrobenzenes influences the kinetic characteristics, apparent K, and V,,, for their conversion by the glutathione S-transferases in a way that can be described by quantitative structure-activity relationships (QSAR's) which are based on outcomes of computer calculations. The use of molecular orbital computer calculations for characterizing a QSAR for the rate of conversion of a series of substrates by glutathione S-transferases provides another approach than the description of QSAR's on the basis of Hammett substituent constants, which is the approach mostly described up to now for the glutathione S-transferase catalyzed reactions.
Introduction The energy of a transition state is a factor influencing the activation energy and, thus, the rate of enzyme catalysis. In addition, relative differences in chemical reactivity of substrates and enzyme cofactors can be expected to be a factor that affects the activation barrier and the conversion of a molecule in the active site of an enzyme. Previous studies on the conversion of 4-hydroxybenzoates by 4-hydroxybenzoate 3-hydroxylase from P s e u d o m o m fluorescens (11, and on the cytochrome P450 catalyzed conversion of a series of aniline derivatives in a iodosobenzene supported microsomal system (21,have demonstrated that outcomes of enzyme catalysis can even *Address correspondence to this author at the Department of Biochemistry,Agricultural University, Dreijenlaan 3,6703 HA Wageningen, The Netherlands. Phone: 31-8370-82868; Fax: 31-8370-84801. Abstract published in Advance ACS Abstracts, April 1, 1995. @
0893-228d95/2708-0481$09.00/0
be predominantly determined by chemical reactivity parameters. These chemical reactivity characteristics of a compound can be quantified on the basis of outcomes of molecular orbital computer calculations. In the present study, two approaches are investigated as a possible way to describe computer calculation based QSAR's for the glutathione S-transferase catalyzed conversion of a series of fluoronitrobenzenes. These approaches are the frontier orbital theory as well as transition state calculations using a model nucleophile (MeS-) representative for the thiolate anion of glutathione. Following frontier orbital theory (3, 4 ) the nucleophilicity of a compound can be quantified by the characteristics of its highest occupied molecular orbital (HOMO) and, if close in energy, of the orbital with energy just below the HOMO, i.e., the HOMO-1. Electrophilicity is dependent on the characteristics of the lowest unoccupied molecular orbital (LUMO) and the LUMO 1. The first approach of the
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482 Chem. Res. Toxicol., Vol. 8, No. 4, 1995
Rietjens et al.
Table 1. 'H and lSF N M R Splitting Patterns of the Aromatic Substituents of the Various Fluoronitrobenzene Glutathione Adductsa compound spectral characteristics "F NMR -106.4 (F4)(mi35F4-H3 = 9.8HZ, 35F4-H5 = 7.4HZ,45F4-H6 = 5.5 HZ) 4-fluoro-2-glutathionylnitrobenzene 'H N M R 7.32(H3j(dd; 3 5 ~ 3 - ~=49.8Hz,4 5 ~ 3 - ~ 5 2.5 Hz);7.01 (H5)(m; 3 5 ~ 5 - ~=47.4Hz, 3 5 ~ 5 - ~=6 9.2HZ,45H5-H3 = 2.5 HZ);8.16 (H6) (dd; 4 5 ~ 6 - ~=4 5.5 Hz, 35H6-H5 = 9.2Hzj I9FNMR: -121.3 (F2)(dd; 3 5 ~ 2 - ~=3 12.2Hz,4JF2-~6 = 8.1 Hz) 2-fluoro-4-glutathionylnitrobenzene 'H NMR: 7.26 (H3j(dd; 3 5 ~ 3 - ~=2 12.2Hz,4 5 ~ 3 - ~=5 2.0Hzj;7.18(H5j(dd; 3 5 ~ 5 - ~=6 8.8 Hz, 45H5-H3 = 2.0HZ);7.95 (H6j(dd; 4 5 ~ 6 - ~=28.1 Hz,45H6-H5= 8.8 Hz) 2,6-difluoro-4-glutathionylnitrobenzene I9FN M R -121.9 (F2/6)(d; 45F216-H3/5= 9.6Hz) 'H NMR: 7.11 (H3/5)(d; 3J~3/5-~216 = 9.6Hz) 4,6-difluoro-2-glutathionylnitrobenzene"F NMR -105.2(F4)(m; 35F4-H3 = 8.9Hz,35F4-H5 = 8.9 HZ,45F4-F6 = 11.7Hz);-119.2 (F6)(dd; 35F6-H3
= 9.8 HZ,45F6-F4 = 11.7 HZ)
'H NMR: 7.24(H3) (dd; 35H3-F4 = 8.9 HZ,45H3-H5 = 2.3 HZ);7.02 (H5)(m;35H5-F6 9.8Hz, 35H6-F4 = 8.9HZ,4 5 H 5 - H 3 = 2.3 HZ) 3,4,6-trifluoro-2-glutathionylnitrobenzene 19FNMR: -126.5 (F6)(m; 3J~6-~5 = 9.2Hz,45F6-F4 = 6.1 Hz,55F6-F3 = 12.2Hz);-129.3 (F4j(m; 35F4-F3 = 17.5 HZ,35F4-H5 = 9.2HZ,45F4-F6 = 6.1HZ);-134.6 (F3)(m; 35F3-F4 = 17.5 HZ, 45F3-H5 = 6.3 HZ,55F3-F6 = 12.2Hzj 'H NMR: 7.37(H5)(In; 35H5-F6 = 9.2HZ,35H5-F4 = 9.2HZ,45H5-F3 = 6.3HZ) 2,3,6-trifluoro-4-glutathionylnitrobenzene I9FNMR: -144.4 (F2)(dd; 3 5 ~ 2 - ~=3 19.9 Hz,4JF2-~6 = 4.8 Hz);-140.3 (F3)(m;35F3-F2 = 19.9Hz, 45F3-H5 = 5.0 HZ,55F3-F6 = 11.7HZ);-126.8 (F6j(m; 35F6-H5 = 11.0 HZ, 4 5 ~ 6 - ~=2 4.8 HZ,5 5 ~ 6 - ~=3 11.7 Hz) 'H N M R : 7.28(H5)(dd, 3 J ~ 5 - = ~ 611.0Hz, 4 5 ~ 5 - ~=3 5.0 Hzj a Chemical shift values are in 2Hz0 and presented relative to CFC13 for 19FNMR and relative to TSP (sodium 3-(trimethylsilyl)(2,2,3,32H4)propionate)for 'H NMR. Difluoronitrobenzene, 2,4,64rifluoronitrobenzene, and 2,3,4,6present study investigates to what extent the frontier tetrafluoronitrobenzene were purchased from Fluorochem (Derorbital parameters for electrophilic attack of the series byshire, U.K.). The corresponding fluorine containing glutathione of fluoronitrobenzenes determine the outcomes of their conjugates were synthesized from 2,4-difluoro-, 2,4,6-trifluoro-, overall conversion by cytosolic glutathione S-transferases. and 2,3,4,6-tetrafluoronitrobenzeneessentially as described The nitrobenzenes used were a series of fluorinated before (11). Spectral characteristics of the compounds were nitrobenzenes, for which the presence of an increasing determined by 19F and lH NMR, and the results, used for number of fluorine substituents can be expected to identification of the various glutathione adducts, are presented influence their electrophilicity. This electrophilicity of in Table 1. the substrates is likely to influence the possibilities for NMR Measurements. Proton decoupled 19FNMR measurea nucleophilic attack on these substrates by the gluments of cytosolic incubations were performed on a Bruker AMX 300 spectrometer as described before (12,13).Between 500 and tathione thiolate anion, known to be the form of the 1000 scans were recorded. The sample volume was 1.75 mL reactive cofactor in the active site of the glutathione containing 100 pL of 2Hz0 for locking the magnetic field and S-transferases (5-7). The fluoronitrobenzenes were also 50 pL of a 8.4 mM 4-fluorobenzoic acid solution, added as chosen because the van der Waals radius of a fluorine internal standard. Concentrations of the various metabolites substituent is only slightly larger than that of a hydrogen could be calculated by comparison of the integrals of the I9F substituent, limiting the influences of sterical hindrance, NMR resonances of the metabolites to the integral of the I9F and, furthermore, because the fluorine substituents make NMR resonance of 4-fluorobenzoic acid. it possible to characterize metabolite patterns using 19F Proton coupled 19FNMR and 'H NMR spectra of synthesized NMR. Previous studies with a series of 4-substitutedglutathione adducts were performed on a Bruker AMX 500 1-chloro-2-nitrobenzeneshave already demonstrated a spectrometer. Upon freeze-drying, the synthesized compounds were dissolved i n 2H20 to give a final sample volume of 0.5 mL. relationship between the logarithm of the rate of conver'H NMR spectra were recorded using 60" pulses (6 p s ) , a 10 sion and the Hammett u values representing the influk H z spectral width, a repetition time of 1.9 s, quadrature phase ence of the C4-substituent on the electrophilicity of the detection, and quadrature phase cycling (CYCLOPS). About 200 substrates (5). However, in studies with 1-chloro-2,4scans were recorded. dinitrobenzene, product release has been demonstrated 'H chemical shift values are presented relative to sodium to be rate-limiting (8-10). The second approach of the 3-(trimethyl~ilyl)(2,2,3,3-~H4)propionate. Proton coupled 19F present study was to base the QSAR on calculations NMR spectra of the Bruker AMX 500 were recorded using a 'H representative for relative changes in the transition state probehead tuned to 470.53 MHz, 30" pulses (3,us), a 15-30 kHz and the intermediates of the reaction. When the formaspectral width, and a repetition time of 1.6 s. tion of the Meisenheimer complex is the rate-limiting step Preparation of Cytosol. Cytosol was prepared from the in the glutathione S-transferase catalyzed conversion of perfused livers of male Wistar rats (400 g) as described before the fluoronitrobenzenes, the relative energy differences (11). Protein content was determined by the method of Lowry e t al. (14) using bovine serum albumin a s the standard. for formation of this complex may be another basis for In Vitro Incubations. Glutathione S-transferase catalyzed QSAR characterization. These relative energies for conversion was studied in cytosolic incubations containing (final formation of the Meisenheimer complex were calculated concentrations) 0.1 M potassium phosphate, pH 7.6, 1 mM using MeS- as the model nucleophile. The use of a model EDTA, 1mM glutathione (reduced form) (Sigma St. Louis, MO), nucleophile will result in absolute values that deviate 0.1-5.0 mg of cytosolic proteirdml (depending on the activity from the heat of formation for the Meisenheimer interwith the respective substrate), and 0.1-20 mM of the nitrobenmediates formed with glutathione. However, the deviazene (depending on the apparent Km),added a s 1% of a 100 tion will be similar for all fluoronitrobenzenes, resulting times concentrated stock solution in dimethyl sulfoxide. The in relative differences between the heat of formation for reaction was started by the addition of the fluoronitrobenzene the Meisenheimer complexes of the various fluoroniand carried out at 37 "C for 5 or 10 min. At 0.5 or 1 min time intervals, 200 pL samples were taken from the incubation trobenzenes that can be compared and used as a basis mixture, mixed with 30 pL, 33%trichloroacetic acid to stop the for QSAR studies. reaction, and assayed for GSH content using Ellman's reagent Materials and Methods (5,5'-dithiobis(2-nitrobenzoic acid)) (Boehringer Mannheim, FRG) (15). From the linear decrease in time of the GSH content, the Chemicals. 2-Fluoronitrobenzene and 4-fluoronitrobenzene glutathione S-transferase catalyzed activity was calculated. were obtained from Aldrich Chemie (Steinheim, Germany). 2,4-
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 483
QSAR's for Glutathione S-Transferase Activities were corrected for GSH decrease due to the chemical reaction between glutathione and the fluoronitrobenzenes, measured in blank incubations carried out in the absence of cytosolic protein. Cytosolic incubations for 19F NMR analysis were similar, containing 1 mg/mL cytosolic protein and 5 mM fluoronitrobenzene. Incubation was at 37 "C, but the incubation time varied with the nitrobenzene derivative, being 1 h for 2,3,4,64etrafluoro- and 2,4,6-trifluoronitrobenzene, 4 h for 2,4difluoronitrobenzene, and 16 h for 2- and 4-fluoronitrobenzene and generally 4 times longer for the incubations in the absence of cytosolic protein. Samples for 19FNMR analysis were stored at -20 "C until analyzed. Chemical reactivity for the QSAR studies were all determined a t 1.5 mM fluoronitrobenzene concentration in 0.1 M potassium phosphate, pH 7.6, 1 mM EDTA, and 1 mM GSH or in 0.1 M sodium pyrophosphate, pH 9.9, 1 mM EDTA, and 1 mM GSH. For these studies time intervals during which samples for GSH determinations were taken varied depending on the rate of the reaction. Time intervals were chosen in such a way that ten samples were taken over the time needed for use of about 20% of the glutathione present. Molecular Orbital Calculations. Molecular orbital calculations were carried out on a Silicon Graphics Indigo using Insight (Biosym, CA). The semiempirical molecular orbital method was used, applying the AMI or the PM3 Hamiltonian from the MOPAC program. Because the results obtained with the PM3 Hamiltonian were in all cases similar to those obtained with the AM1, only the results of the AM1 calculations are presented. All calculations were carried out with PRECISE criteria. For all calculations the self-consistent field was achieved. Geometries were optimized for all bond lengths, bond angles, and torsion angles using the BFGS criteria. Frontier electron densities to characterize electrophilicitywere calculated from LUMO (lowest unoccupied molecular orbital) and LUMO 1characteristics using the equation given by Fukui et al. (4). In this study, the outcomes of the semiempirical calculations on molecules in vacuum are related to the electronic characteristics of the substrates in the active site of the glutathione S-transferases. Due to solvation effects and a different dielectric constant, the intrinsic properties of the compounds might be influenced upon binding to this active site. However, it is assumed t h a t this phenomenon will not influence the relative differences of parameters between a series of closely related compounds or between similar centers within one molecule, to a significant extent. The outcomes of the in vacuo computer calculations can thus be used as an approach to study relative differences within a series of related compounds U , 2 )or within one molecule (13). Relative heats of formation, Le., AAHF values, were calculated as follows. Using the AM1 (or PM3) Hamiltonian the heats of formation for the various fluoronitrobenzenes as well as for the various MeS- Meisenheimer complexes were calculated. The heat of formation of a fluoronitrobenzene was then subtracted from the heat of formation for its Meisenheimer MeS- complex, leading to a AHF value. The AHF values thus obtained were set to a relative scale in which the lowest AHF was set to zero, thus providing the relative AHF, Le., the AAHF values. The AAHF values, calculated from the computer programs in kcal/mol, were converted to eV to provide values in the same units as used for the E(LUM0). For 2,4-difluoro-, 2,4,6-trifluoro-, and 2,3,4,6-tetrafluoronitrobenzene, more than one Meisenheimer complex can be formed. For these compounds a weighted average AAHF was calculated, using a weight factor for the various Meisenheimer complexes that was based on the regioselectivity observed for the glutathione conjugation (see Table 2 in the Results section). Calculation of Log Pootanol. Log Paetanol values were calculated as described by Rekker and de Kort (16).
a
F
F'
IS 1
Conversion of Fluoronitrobenzenes by Cytosolic GlutathioneS-Transferases. Figure 1 p r e s e n t s the 19F NMR s p e c t r a of cytosolic i n c u b a t i o n s of t h e fluoroni-
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Figure 1. I9F NMR spectra of cytosolic incubations with GSH and (a) 2-fluoronitrobenzene, (b) 4-fluoronitrobenzene, (c) 2,4difluoronitrobenzene, (d) 2,4,64rifluoronitrobenzene, and (e) 2,3,4,64etrafluoronitrobenzene.Metabolites were identified by the results presented in Table 1. The third resonance of 2,3,6trifluoro-4-glutathionylnitrobenzeneat -126.8 ppm (panel e) is masked by one of the resonances of the parent compound. t r o b e n z e n e s in the presence of g l u t a t h i o n e . F o r 2-fluoronitrobenzene and for 4-fluoronitrobenzene (Figure la,b)
Rietjens et al.
484 Chem. Res. Toxicol., Vol. 8, No. 4, 1995
Table 2. Regioselectivity of the Chemical and Glutathione S-TransferaseCatalyzed Glutathione Conjugation of the Fluorinated Nitrobenzene As Determined by leF NMRa relative percentage of glutathione conjugation nitrobenzene
conjugation at center
chemical reaction, pH 7.6
chemical reaction, pH 9.9
enzymatic reaction
2-fluor0 4-flu0r 0 2,4-difluoro 2,4,6-trifluoro 2,3,4,6-tetrafluoro
CLC2 CLC4 Cl:C2:C4 Cl:C2/6:C4 Cl:C2:C3:C4:C6
0:lOO 0:lOO 0:91:9 0:94:6 0:90:0:10:0
0:lOO 0:lOO 0:92:8 0:98:2 0:99:0:1:0
0:lOO 0:100 0:63:37 0:78:22 0:70:0:30:0
a Values presented are the mean of two independent incubations, the results of which vary by 5 1%.The regioselectivity of the chemical reaction was determined at pH 7.6 (pH of the enzymatic incubations) but also a t pH 9.9 where the GSH becomes deprotonated as in the active site of the glutathione S-transferases (5-7). Only nitro- or fluorine-substituted sites are taken into consideration.
formation of fluoride anions is observed, indicating that glutathione conjugation occurs at the fluorinated position, resulting in fluoride anion elimination upon formation of the adduct. Elimination of the nitro moiety, resulting respectively in (2-fluoropheny1)glutathioneand (4-fluorophenyl)glutathione, is not observed, as in addition to the parent and fluoride anion peak no other fluorine resonances are observed in the spectra. For 2,4-difluoronitrobenzene (Figure IC)formation of two metabolites is observed, which, based on their splitting patterns in I9F and 'H NMR (Table 11, can be ascribed to 4-fluoro2-glutathionylnitrobenzeneand 2-fluoro-4-glutathionylnitrobenzene. The two metabolites, resulting from glutathione conjugation at C2 and C4 of 2,4-difluoronitrobenzene, are formed in a ratio of 0.63:0.37, the reaction at C2 being favored over a reaction a t C4. The formation of the two glutathione adducts is accompanied by formation of an amount of fluoride anions that equals the sum of formation of the two defluorinated glutathione conjugates. Conversion of 2,4,6-trifluoronitrobenzene (Figure Id) results in formation of 4,6-difluoro-2-glutathionylnitrobenzene and 2,6-difluoro-4-glutathionylnitrobenzene at a ratio of 0.78:0.22. Apparently, as for 2,4difluoronitrobenzene,formation of the metabolite resulting from conjugation at the position(s) ortho with respect to the nitrosubstituted carbon center is favored over the one resulting from conjugation at the halogenated para position. The formation of the two glutathione adducts is accompanied by formation of an amount of fluoride anions that equals the sum of formation of the two defluorinated glutathione conjugates. Finally, Figure l e presents the I9FNMR spectrum of a cytosolic incubation with 2,3,4,6-tetrafluoronitrobenzene, in which formation of 3,4,6-trifluoro-2-glutathionylnitrobenzene and 2,3,6-trifluoro-4-glutathionylnitrobenzene can be observed at a ratio of 0.70:0.30, accompanied by formation of a corresponding amount of fluoride anions. Formation of 2,3,4-trifluoronitro-6-glutathionylnitrobenzene and of 2,4,6-trifluoro-3-glutathionylnitrobenzene is not observed. Furthermore, as for the other fluoronitrobenzenes, formation of the adduct resulting from substitution of the nitro moiety is not observed. Table 2 summarizes the regioselectivities thus obtained for the conjugation of the various fluoronitrobenzenes by the cytosolic glutathione S-transferases. The table also presents results for the regioselectivities of the reaction observed in the absence of cytosol, both at pH 7.6 and at pH 9.9, a pH at which the glutatione becomes deprotonated to give the thiolate anion. From the data obtained it follows that in a chemical assay a t pH 7.6, but also at pH 9.9, the preferential site for glutathione conjugation of a fluorinated nitrobenzene is at the positions ortho with respect to the nitro moiety. The fluorinated ortho C2 position in the polyfluoronitrobenzenes becomes sub-
Table 3. Apparent V,, and Km Values for the Conversion of Fluoronitrobenzenesby Cytosolic Glutathione S-Transferasesat 1 mM GSH" V,,
[nmol of GSH reacted-min-'. (mg of protein)-I] K, (mM)
nitrobenzene 2-fluoronitrobenzene 4-fluoronitrobenzene 2,4-difluoronitrobenzene 2,4,6-trifluoronitrobenzene 2,3,4,6-tetrafluoronitrobenzene
6.3 f 0.7 7.6 i 1.7 108 f 9 414 & 43 1661 f 535
3.8 f 0.7 3.8 f 0.5 2.4 f 0.8 1.5 f 0.5 0.4 f 0.3
Values presented are the mean f SEM ( n = 2-3).
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log Poctanol Figure 2. Relation between the apparent K, for conversion of a series of fluorinated nitrobenzenes by cytosolic glutathione The correlation coefficient S-transferases and their log Poctanol. of the QSAR is -0.997.
stituted to an about 9 times higher extent than the fluorinated para C4 position. However, the reaction catalyzed by the cytosolic glutathione S-transferase shows a lower extent of regioselectivity: the ortho conjugation is favored over para conjugation by a factor of about 2.5-3.5, pointing to an enzyme mediated effect on the regioselectivity. Surprisingly, for 2,3,4,6-tetrafluoronitrobenzene a strong preference for the fluorinated C2 over the fluorinated C6 ortho position is observed in both the enzymatic as well as the chemical glutathione conjugation. Kinetic Characteristics of the Conversion of the Fluoronitrobenzenes by Cytosolic Glutathione STransferases. Table 3 presents the results from kinetic experiments characterizing the apparent V, and K , for the fluoronitrobenzene compounds in cytosolic incubations with GSH. From the data obtained it can be concluded that the apparent K, decreases whereas the apparent V, rises substantially with increasing number of fluorine substituents. Figure 2 shows that this change in K, correlates quantitatively ( r = -0.997) with the log Poctanol for the fluoronitrobenenes, indicating that the
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 486
QSAR's for Glutathione S-Transferase Table 4. Electrophilic Characteristics of the Fluoronitrobenzenes Calculated with the AM1 Hamiltoniana
a
LUMO density at carbon center
nitrobenzene 2-fluor0 4-flUOrO 2,4-difluoro 2,4,6-trifluoro 2,3,4,64etrafluoro
E(LUM0) rest of (eV) C 1 C2 C3 C4 C5 C6 moleculeb -1.30 0.20 0.16 0.02 0.24 0.04 0.11 0.23 -1.35 0.19 0.12 0.03 0.26 0.03 0.12 0.25 -1.58 0.19 0.15 0.03 0.27 0.04 0.10 0.22 -1.81 0.20 0.13 0.03 0.27 0.03 0.13 0.21 -2.07 0.20 0.13 0.04 0.28 0.04 0.13 0.18
'?
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The PM3 Hamiltonian gave similar results. The total LUMO density on the molecule should equal 1 ( 4 ) .
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apparent K, values decrease upon increased hydrophobicity (increased log Poctanol) of the fluoronitrobenzene substrates. Molecular Orbital Substrate Characteristics for Electrophilic Attack. Table 4 presents the calculated characteristics of the fluorinated nitrobenzene derivatives of importance for their reactivity in an electrophilic attack on the glutathione thiolate anion, known to be present in the active site of the glutathione S-transferases (5-7). Because a thiolate anion is generally accepted to be a so-called soft nucleophile, it can be expected that frontier orbital characteristics will be of major importance for the possibilities for its reactivity with a substrate (3). The substrate characteristics of importance for electrophilic reactivity are the energy of the lowest unoccupied molecular orbital, Le., the orbital that will interact with the most reactive electrons in the highest occupied molecular orbital of the glutathione thiolate anion, and the density of the LUMO on the respective reaction centers. Because the LUMO 1 is generally 0.7eV higher in energy than the LUMO itself, 1 to the density the contribution of the LUMO distribution for electrophilic attack, calculated as described by Fukui et al. (41, appears neglegible (data not shown). The results from the calculations (Table 4) demonstrate a decrease in the energy of the LUMO with increasing number of fluorine substituents, pointing a t increased electrophilic reactivity of the nitrobenzenes with increased number of fluorine substituents. Furthermore, the highest density for a nucleophilic attack on the fluoronitrobenzenes is genrally located at the nitro substituted C1, the ortho C2 and C6, and the para C4 positions, the C3 and C5 centers being less reactive. Comparison of the differences in calculated reactivity for an electrophilic attack of the various fluorinated carbon centers (Table 4) to the actual regioselectivity of the enzymatic glutathione conjugation (Table 2) demonstrates that the actual site of conjugation does not correlate with the calculated reactivity of the various positions. Only the low reactivity of the C3 position in 2,3,4,6-tetrafluoronitrobenzenecan be explained by the low electrophilic reactivity at this carbon center reflected by a low LUMO density. The same conclusion holds for the regioselectivity observed for the chemical reaction, both at pH 7.6and at pH 9.9.The regioselectivity in the chemical reaction even further deviates from the calculated differences in electrophilic reactivity between the various reaction centers within the fluoronitrobenzenes. From the results in Table 4 it can also be derived that the relative densities at the corresponding fluorinated carbon centers in the different fluoronitrobenzenes are similar. This indicates that a relative difference in density of the reactive LUMO orbital on these reaction sites cannot account for the more than 200-fold change in apparent V,, for the conversion of the various
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1 0.5 0.6 a 7
AAHF in eV Figure 3. QSAR's describing the quantitative structureactivity relationships between the natural logarithm of the apparent V, for cytosolic glutathione 5'-transferase catalyzed conjugation of a series of fluorinated nitrobenzenes and (a) the E(LUM0) of the compounds and (b) the relative AAHF for formation of the MeS- Meisenheimer complex. The correlation coefficients are -0.986 (panel a) and -0.987 (panel b). Theoretically, V, should be divided by hlkT to make the parameter dimensionless before calculating the natural logarithm. However, because this would result in a change of the y-axis values by a constant factor, this theoretically appropriate correction was omitted and the natural logarithm of V,, was plotted.
fluorinated nitrobenzenes. However, Figure 3, in which the natural logarithm of the V,, is plotted against the calculated energy of the LUMO, shows a clear correlation (r = -0.986, AM1 Hamiltonian) between these two parameters. With the E(LUM0) values calculated by the PM3 Hamiltonian a similar correlation (r = -0.985)is obtained. Thus, in the present study a molecular orbital based QSAR (MO-QSAR)is obtained for the overall rates of conversion of the series of fluoronitrobenzenes by cytosolic glutathione S-transferases. Relative Heats of Formation (AAHF)for Formation of the Meisenheimer Complexes. Table 5 presents the AAHF values calculated for formation of the various Meisenheimer complexes of the five fluoronitrobenzenes as well as the weighted average AAHF values. The weight factor was based on the regioselectivity of the reaction presented in Table 2. Figure 3b presents the QSAR obtained when for the enzymatic reaction the AAHF is plotted against the natural logarithm of the Vma. The correlation coefficient of the QSAR is -0.987. On the basis of the QSAR presented in Figure 3b and the actual AAHF values obtained for the formation of the possible Meisenheimer complexes from either 2,4-difluoro-, 2,4,6-trifluoro-, and 2,3,4,6-tetrafluoronitrobenzene (Table 5 ) , one can calculate the relative rates of
486 Chem. Res. Toxicol., Vol. 8, No. 4,1995
Rietjens et al.
Table 5. Relative Heats of Formation (AAHF) for the Formation of the Meisenheimer Complexes of the Fluoronitrobenzenes with MeS- as the Model Nucleophile, and the Weighted Average Valuesa MeS- Meisenheimer AAHF AAHF weighted average AAHF (eV) fluoronitrobenzene complex a t carbon center (kcal/mol) (eV) enzyme catalyzed chemical reaction, pH 7.6 chemical reaction, pH 9.9 2-fluor0 c2 15.4 0.668 0.668 0.668 0.668 4-fluor0 c4 13.7 0.594 0.594 0.594 0.594 2,4-difluoro c2 10.2 0.440 0.422 0.436 0.436 c4 9.0 0.391 2,4,6-trifluoro C2/6 4.8 0.207 0.204 0.206 0.207 c4 4.4 0.193 2,3,4,64etrafluoro c2 1.1 0.048 0.036 0.044 0.048 c3 13.7 0.595 c4 0.2 0.008 C6 0 0 a
The weight factor was based on the regioselectivity of the glutathione conjugation presented in Table 2.
reaction expected for conjugation at the various fluorinated centers. On the basis of the rates of reaction thus calculated for the different fluorinated centers in a fluoronitrobenzene, one might predict the regioselectivity of the glutathione conjugation. For example, for 2,4difluoronitrobenzene the QSAR in Figure 3b and the AAHF values presented in Table 5 give rise to calculated rates of reaction that predict a regioselectivity of 0.39: 0.61 for conjugation at C2 and C4. In a similar way, for 2,4,6-trifloronitrobenzene a C2/6:C4 ratio of 0.64:0.36 and for 2,3,4,6-tetrafluoronitrobenzenea C2:C3:C4:C6 of 0.25: 0:0.36:0.39 is calculated. Comparison of these predicted values to the actual regioselectivities observed (Table 2) indicates that the AAHF approach also cannot explain the regioselectivity of the glutathione conjugation. QSAR's for the Chemical Reaction of Glutathione with the Fluoronitrobenzenes. For comparison with the QSAR's obtained for the enzymatic reaction (Figure 31, the rates of the chemical reaction of the fluoronitrobenzenes with glutathione, both at pH 7.6 and at pH 9.9, were determined. This was done at a fixed nitrobenzene concentration of 1.5 mM. Figure 4 presents the QSAR's thus obtained using either E(LUM0) of the fluoronitrobenzenes or the AAHF as a parameter for the QSAR. The QSAR's for the enzymatic reaction are also presented. From the data presented it can be derived that the rate of reaction at pH 9.9 is always higher than at 7.6, which can be explained by the fact that deprotonation of glutathione to the thiolate anion will activate its nucleophilicity. The fact that the QSAR's obtained for the chemical reaction at pH 7.6 and 9.9 are parallel indicates that the rate enhancement observed upon deprotonation of the glutathione is by the same factor for all fluoronitrobenzenes. Figure 4 also demonstrates that the QSAR's obtained for the chemical reactions are parallel to the QSAR obtained for the enzymatic reaction. Assuming that 1-5% of the cytosolic protein actually consists of glutathione S-transferases, and taking into account the molecular weight of the various isoenzyme monomers, the V,, values (expressed in nmol of GSH reacted.min-l.(mg of protein)-l) can be converted to a first order rate constant in min-'. The rate for the chemical reaction (in pmol of GSH reactedmin-'(L of incubation)-') measured at 1 mM GSH and 1.5 mM nitrobenzene can be converted to a second order rate constant in min-l-M-'. The effective molarity (in M) that can be calculated as the ratio of the first order and second order rate constants thus obtained is 100-400 for the glutathione S-transferase catalyzed reaction at pH 7.6. This effective molarity gives an impression of the relative rate enhancement by the enzyme compared to the chemical reaction at pH 7.6. The fact that the QSAR's of the chemical and enzymatic reaction are parallel and independent of the
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A A H F in eV Figure 4. QSAR's for the enzymatic and the chemical conjugation of glutathione with the series of fluoronitrobenzenes with (a) E(LUM0) of the fluoronitrobenzene and (b) AAHF for formation of the MeS- Meisenheimer complex as the parameter for the QSAR. The QSAR's present the correlations for ( 0 )the enzymatic reaction, (A)the chemical reaction at pH 9.9, and (+) the chemical reaction at 7.6. Correlation coefficients were at least -0.97 in all cases. For the enzymatic reaction V,, is in nmol of GSH reacted.min-l.(mg of protein)-l; for both chemical reactions Vis in pmol of GSH reactedmin-YL of incubation)-'. The chemical reaction rate is dependent on the concentration of both substrates and was carried out at fixed concentrations of glutathione (1mM) and fluoronitrobenzene (1.5 mM). In fact, instead of In V the value of In k could have been given, which would change the scale by a constant factor. Theoretically, V, and V values should be dimensionless before calculating the natural logarithm. However, because the respective corrections would result in a change of the y-axis values by a constant factor, this theoretically appropriate correction was omitted and the natural logarithm of V,, and V was plotted.
above-mentioned factors indicates that in the enzymatic reaction chemical reactivity parameters determine the overal outcome of catalysis and, in addition, that the chemical and enzymatic reactions proceed through a similar reaction pathway with comparable reaction in-
QSAR's for Glutathione S-Transferase
termediates.
Discussion The glutathione S-transferase catalyzed conversion of halogenated nitrobenzenes is known to proceed by an aromatic nucleophilic substitution reaction which proceeds through formation of a so-called a or Meisenheimer complex intermediate (5, 8, 9). Previous studies with substituted 1-chloro-2-nitrobenzenes have already demonstrated that formation of this a complex might be the rate-limiting step in overall catalysis (5). For specific substrates another step might become rate-limiting. For instance, the presence of the second nitro group in l-chloro-2,4-dinitrobenzenehas been reported to result in product release as the rate-limiting factor in its conversion by isoenzyme 3-3, perhaps due to additional possibilities for hydrogen bonding of the reaction product with the second nitro moiety (8-10). However, for a series of substituted mononitrobenzenes, the rate-limiting character of the initial nucleophilic attack of the glutathione cofactor on the aromatic substrate, leading to formation of the a complex, is reflected in clear correlations between Hammett a values and the logarithm of the maximal turnover rate for both the chemical (17) as well as the enzymatic (5) conversion. These correlations with Hammett CJ substituent constants clearly demonstrate the increased possibilities for glutathione conjugation with increased electrophilicity of the substrates, The results of the present study with a series of fluorinated nitrobenzenes demonstrate that the electrophilicity of a substrate and, thus, its overall reactivity in a glutathione S-transfersae catalyzed conjugation reaction can also be characterized by a calculated molecular orbital parameter. For the series of fluoronitrobenzenes, with increasing number of fluorine substituents, the natural logarithm of their apparent V, in a reaction catalyzed by cytosolic glutathione S-transferases correlates with the calculated energy of their lowest unoccupied molecular orbital (E(LUM0))( r = -0.986). The rate of the overall reaction increases when the E(LUM0) of the fluoronitrobenzene decreases. This indicates, following frontier orbital theory ( 3 , 4 ) ,that the possibilities for an efficient orbital interaction between the LUMO of the fluorinated nitrobenzene substrate and the HOMO of the glutathione thiolate anion increase, upon lowering the E(LUM0) of the substrate. Increased possibilities for efficient orbital interaction, resulting from a decreased energy gap between the two interacting orbitals (31, provide increased possibilities to overcome the activation barrier of a reaction. Preliminary results from V,, determinations for the conjugation of 2-chloronitrobenzene, 2-fluoro-5-aminonitrobenzene, and 2fluoro-5-methylnitrobenzene, with E(LUM0) values of respectively -1.07, -1.18, and -1.25 eV, show that the Q S A R presented in Figure 3a can be extended to nitrobenzene derivatives with other than fluorine substituents (correlation coefficient = 0.992). However, a full investigation of the possibilities to extend the MO-QSAR approach to other substrates than the fluoronitrobenzenes of the present study is presently undertaken. A second approach to describe a QSAR on the basis of computer calculated parameters was to use the relative AAHF for formation of the Meisenheimer transition state of the reaction. The present study shows that this approach provides a good QSAR correlation €or the rate of conjugation of the fluoronitrobenzenes, but is also unable to explain the regioselectivity of the glutathione conjugation.
Chem. Res. Toxicol., Vol. 8, No. 4,1995 487 The QSAR's described in the present study corroborate the conclusion derived before in the literature for other types of halogenated mononitrobenzenes (5) that the initial attack of the glutathione thiolate anion on the fluoronitrobenzene, resulting in formation of the a complex, represents the rate-limiting step in overall catalysis. The observation that the QSAR's for the chemical conjugations are parallel to the QSAR's for the enzymatic reaction further supports this conclusion. The two QSAR approaches of the present study, i.e., based on frontier orbital theory and on transition state calculations, demonstrate the importance of chemical reactivity for the rate limitation of glutathione S-transferase catalyzed conversion for a series of fluoronitrobenzenes. This implies that both these approaches provide a way to describe QSAR's for substrates with increasing number of (halogen) substituents, instead of for a Hammett series of substrates with variation in one substituent at a specific position in the molecule. In addition, the results of the present study demonstrate a Q S A R for the apparent K, of the conversion of the fluoronitrobenzenes by the cytosolic glutathione S-transferases. Although a K, is dependent on additional kinetic constants than the ones determining the dissociation constant between the enzyme and its substrate, Le., &, the relationship between log Poctanol and the apparent K, could reflect increased affinity of the glutathione S-transferases for fluoronitrobenzenes with increased hydrophobicity. Finally, it must be stressed that the computer calculation based QSAR's and the regioselectivity results of the present study are obtained with a mixture of cytosolic glutathione S-transferases in which the contribution of the various isoenzymes to the overall effect may vary. Isoenzyme 3-3, for example, might be a better catalyst for the nucleophilic aromatic conjugation than other isoenzymes (10,18). However, to what extent the regioselectivity observed in the cytosolic mixture of glutathione 5'-transferases originates from a highly regioselective contribution of different isoenzymes, some specifically conjugating at C2 while others at C4,or whether various isoenzymes all show a mixed regioselectivity, is an interesting topic for future investigations, but beyond the scope of the present paper. This matter, as well as computer calculation based QSAR studies with isolated glutathione S-transferases, providing possibilities for comparison of the slope of the MO-QSAR obtained for different glutathione S-transferases, are presently under investigation. Altogether, the results of the present study clearly demonstrate that the number of fluorine substituents in the fluoronitrobenzene model compounds influences the kinetic characteristics, apparent K, and VmU, for their conversion by the glutathione S-transferases in a way that can be described in quantitative structure- activity relationships which are based on parameters from computer calculations. This computer calculation based approach for characterizing a QSAR for the rate of conversion of a series of substrates by glutathione Stransferases provides another approach than the description of QSAR's on the basis of Hammett substituent constants, which is the approach mostly described up to now for the glutathione S-transferase catalyzed reactions.
References (1) Vervoort, J., Rietjens, I. M. C. M., Van Berkel, W. J. H., and Veeger, C. (1992) Frontier orbital study on the 4-hydroxybenzoate-
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