Biochemistry 1988, 27, 647-654
647
Dissection of the Catalytic Mechanism of Isozyme 4-4 of Glutathione S-Transferase with Alternative Substrates? Wen-Jian Chen, Gerard F. Graminski, and Richard N. Armstrong* Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742 Received July 31, 1987; Revised Manuscript Received September 17, 1987
ABSTRACT: The kinetic and chemical mechanism of isozyme 4-4 of rat liver glutathione (GSH) S-transferase was investigated by using several alternative peptide substrates including N-acetyl-GSH, y-L-glutamyl-Lcysteine (y-GluCys), ~-(malonyl-~-cysteinyl)-~-2,4-diaminobutyrate (retro-GSH), and N4-(N-acetyl-~cysteinyl)-~-2,4-diaminobutyrate(decarboxylated retro-GSH). The enzyme, which is normally stereospecific in the addition of G S H to the oxirane carbon of R absolute configuration in arene oxide substrates, loses its stereospecificity toward phenanthrene 9,lO-oxide with the retro peptide analogues, giving a 2: 1 mixture of the S,S and R,R stereoisomeric 9,1O-dihydro-9-(S-peptidyl)-10-hydroxyphenanthrenes. The analogues with normal peptide bonds (N-acetyl-GSH and y-GluCys) show normal stereospecific addition. The kinetic mechanism of the enzyme was investigated by using the alternative substrate diagnostic with several 4substituted 1-chloro-2-nitrobenzenes and G S H , N-acetyl-GSH, and y-GluCys. Varying the concentration of electrophile vs the identity of the G S H analogue and the concentration of G S H vs the identity of the electrophile gave two sets of intersecting reciprocal plots, a result consistent with a random sequential kinetic mechanism. The pH profiles of k, and k,/K", [saturating G S H , variable 1-chloro-2,4-dinitrobenzene(l)] exhibit a dependence on a deprotonation in the enzyme-GSH-1 and enzyme-GSH complexes with molecular pKa's of 6.1 and 6.6, respectively. This result is consistent with spectroscopic studies that suggest the pKa of the thiol of bound G S H is 1 6 . 8 . In similar experiments with fixed, saturating 1 and variable peptide, kinetic pKa's and limiting turnover numbers at high pH (k:") of 6.1 (2.5 s-l), 7.5 (0.33 s-'), and 7.7 (0.10 s-l) were obtained from plots of log k, vs pH for G S H , N-acetyl-GSH, and y-GluCys, respectively. A plot of log k:m vs the apparent pKa of the nucleophile gave an anomalous Bransted &,,(obsd) = -0.79 f 0.15. In contrast, the specific-base-catalyzed reaction exhibited a p,,, = 0.16 f 0.02 for thiolate anions with conjugate acid pKa's in the range of 7.3-9.5. The anomalous P,,,(obsd) for the enzyme-catalyzed reaction is interpreted to suggest that the p R s observed in pH-rate profiles of k, for N-acetyl-GSH and y-GluCys do not reflect the ionization of the peptide thiol in the ternary complexes of the alternative substrates. Hammett plots of log k, vs u- for a series of 4-substituted 1-chloro-2-nitrobenzenesgive p values of 1.2, 1.9, and 2.5 for G S H , N-acetyl-GSH, and y-GluCys, respectively, as compared to 3.4 for the specificbase-catalyzed reaction with G S H . The results indicate the importance of the peptide structure for the correct orientation of the thiol in the active site and suggest that one major contribution of the enzyme to catalysis is to lower the pKa of the bound nucleophile. Finally, 4-fluoro-3-nitro- 1-(trifluoromethy1)benzene was found to be a much better substrate than the 4-chloro substrate in both the specific-base- and enzyme-catalyzed reactions. This observation suggests that both reactions have similar rate-determining steps, the formation of a a-complex intermediate.
combinations of 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 (Mannervik & Jensson, 1982; Jakoby et al., 1984). Even though the enzyme has received considerable attention in the last several years, relatively little is known concerning the details of its catalytic mechanism. Isozymes 1-1 and 3-3 have been characterized with respect to their kinetic mechanisms. Initial velocity and product inhibition studies of iso-
T e glutathione S-transferases (EC 2.5.1.18) are a group of enzymes that function primarily as catalysts in the nucleophilic addition of the thiol of glutathione (GSH)' to molecules containing electrophilic functional groups. In doing so they act as catalytic scavengers of potentially toxic substances (Jakoby & Habig, 1980). The importance of this family of proteins is most clearly evinced by their high concentrations in liver tissue. Recent reviews are available (Jakoby & Habig, 1980; Mannervik, 1985; Armstrong, 1987). The GSH transferases most thoroughly studied with respect to structure and mechanism are those isolated from rat liver cytosol. The enzymes are homo- or heterodimeric proteins with subunit types designated 1, 2, 3, 4, 5, 6, etc. (Jakoby et al., 1984). Although there is considerable evidence for microheterogeneity in subunit types (Tu & Reddy, 1985; Satoh et al., 1985; Rothkoph et al., 1986), the six major isozymes in rat liver appear to occur in two families with binary subunit
Abbreviations: GSH,glutathione; rGSH, retro-glutathione [N4(malonyl-~-cysteinyl)-~-2,4-diaminobutyrate]; dcrGSH, N4-(N-acetyl~-cysteinyl)-r-2,4-diaminobutyrate; NAcGSH, N-acetylglutathione; yGluCys, y-L-glutamyl-L-cysteine; CysOEt, L-cysteine ethyl ester; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); MES, 2-(N-morpholino)ethanesulfonic acid; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, 3-(N-morpholino)propanesulfonic acid; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane; TAPS, 3- [ [tris(hydroxymethyl)methyl]amino]propanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CAPS, 3-(cyclo-
'This work was supported by N I H Grant G M 30910. *Address correspondence to this author. Recipient of a National Institutes of Health Research Career Development Award (ES 00133).
hexylamin0)- 1-propanesulfonic acid; DSS, 2,2-dimethyl-2-silapentane5-sulfonate; Michaelis constant for G S H or analogues; K",, Michaelis constant for electrophilic substrate; SDS, sodium dodecyl sulfate; HPLC, high-performance liquid chromatography; CD, circular dichroism.
0006-296018 8 10427-0647$01.50/0
eptide,
0 1988 American Chemical Society
648
B I 0 cH E M I sT R Y
C H E N E T .4L.
Chart I
Chart 11
\
SH
X I. SH
I 7
J
5 (3
SH
zymes 1-1 (Schramm et al., 1984) and 3-3 (Jakobson et al., 1977, 1979a,b) clearly suggest random sequential mechanisms for both isozymes. Dead-end inhibition patterns of isozyme 3-3 by the oxygen and dethio analogues of GSH are also consistent with a random sequential mechanism (Chen et al., 1985). Less is understood about the chemical mechanism of catalysis. It is certainly reasonable to assume and some experimental evidence, with different electrophilic substrates, suggests that the protein enhances the nucleophilic reactivity of the thiol of bound G S H relative to that free in solution (Keen et al., 1976; Jakoby & Habig, 1980). In principle, the enzyme could accomplish this by increasing either the fraction of nucleophile (thiolate anion) or its nucleophilicity or both when G S H is bound to the protein surface. Of the rat liver enzymes, isozyme 4-4 has been shown to be unique in its catalytic efficiency and stereoselectivity toward arene oxide substrates (Cobb et al., 1983; Boehlert & Armstrong, 1984; deSmidt et al., 1987). However, essentially nothing is known concerning its kinetic or chemical mechanism of catalysis. Dead-end inhibition studies are consistent with either a random sequential mechanism or an ordered mechanism with GSH adding first (Chen et al., 1985). Preliminary spectroscopic evidence indicates that the pKa of the thiol of G S H bound to isozyme 4-4 is 1 6 . 8 or at least 2.0 pKa units below that in aqueous solution (Graminski et al., 1987). The effect of this apparent shift in pKa of the bound thiol on the nucleophilicity of the thiolate is not clear. The use of alternative substrates is an attractive approach for the investigation of both the kinetic and chemical mechanisms of catalysis of isozyme 4-4. In the first instance, it has been demonstrated by Northrop (Radika & Northrop, 1984) that alternative substrates provide a simple diagnostic tool for distinguishing ping-pong, Theorell-Chance, ordered, and ranodm mechanisms for two-substrate enzymic reactions. Second, manipulation of the electronic character of the electrophilic substrate can yield, in principle, from linear free energy relationships, information on the rate-limiting step(s) and transition state of the reaction. In addition, it is reasonable to expect that the enhancement of the nucleophilic reactivity of glutathione by the enzyme must involve specific recognition of the tripeptide by the protein surface and the utilization of intrinsic binding energy. Therefore, synthetic alterations of the tripeptide structure should provide an indirect method for
X
CI
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CI
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changing the nucleophilicity (or pKa) of the putative enzyme-bound thiolate. In this paper we report the use of four peptide analogues of G S H (see Chart I) and several 4-substituted l-halo-2nitrobenzenes (see Chart 11) in an investigation of the catalytic mechanism of isozyme 4-4 of rat liver GSH transferase in nucleophilic aromatic substitution reactions. The results indicate that the enzyme follows a random sequential kinetic mechanism. Structural alterations of the peptide substrate appear to affect both the effective pK, of the bound thiol and the relative orientation of the thiol with respect to the electrophilic substrate. Substituent and leaving group effects on k, and k,/KS, for substituted halonitrobenzenes are consistent with a mechanism involving rates limiting or partially rate limiting formation of an enzyme-bound u- or Meisenheimercomplex intermediate between glutathione and the haloarene.
EXPERIMENTAL PROCEDURES Materials Enzyme. Isozyme 4-4 of rat liver G S H transferase was purified by the following modification of previously described procedures. Approximately 160-g male Wistar rat livers (Pel-Freez) were homogenized, centrifuged, and chromatographed on a 5 X 55 cm DEAE-cellulose column (Whatman DE-52) as described by Habig et al. (1974). Most GSH transferases, except isozyme 4-4, were eluted from the column with 10 mM Tris-HC1 (pH 8.0). Isozyme 4-4 was eluted from the column with the same buffer containing 0.1 M KC1. This fraction was further purified by precipitation with 96% saturated ammonium sulfate, dialysis, and chromatography on an S-hexylglutathione affinity matrix (Guthenberg & Mannervik, 1979). Finally, the protein was chromatographed on a 2 X 50 cm bed of hdyroxylapatite equilibrated with 10 mM K H 2 P 0 4(pH 6.7) and eluted with a 2-L linear gradient of 10-300 mM KH,P04 (pH 6.7). Isozyme 4-4 was concentrated to about 2 mg/mL, dialyzed against 10 mM K H 2 P 0 4 (pH 6.7), and flash frozen for storage at -80 O C . The purified protein was judged to be 295% homogeneous by the criteria of SDS gel electrophoresis and high-performance anion-exchange chromatography on a 0.5 X 5 cm Pharmacia Mono-Q column eluted at 1 mL/min with 10 mM Tris-HC1, pH 8.0, for 2 min followed by a linear gradient of 0-0.4 M NaCl in 30 min. Retention time for isozyme 4-4 was 5.6 min. Identity of the enzyme was confirmed by amino-terminal sequence analysis, which gave
PMTLGYWDIRGLAHAIRLFLEYTDTSYEDKKYSMGDAPDY?R, a sequence consistent with that known for isozyme 4-4 (Mannervik, 1985; &in et al., 1986). Electrophilic Substrates. Phenanthrene 9,lO-oxide was prepared by the method of Krishnan et al. (1977). 1-
CATALYTIC MECHANISM OF GLUTATHIONE S-TRANSFERASE
Chloro-2,4-dinitrobenzene(1) and 4-chloro-3-nitrophenyl methyl sulfone (2) were from Eastman and ICN Biochemicals, respectively. 4-Chloro-3-nitroacetophenone(3), 4-chloro-3nitrobenzaldehyde (4), 4-chloro-3-nitro- 1-(trifluoromethyl)benzene (5), 4-chloro-3-nitrobenzenesulfonicacid ( 6 ) , and 4-fluoro-3-nitro-1-(trifluoromethyl)benzene (7) were obtained from Aldrich. Glutathione Analogues. retro-Glutathione (rGSH) and decarboxylated retro-glutathione (dcrGSH) were synthesized as described earlier (Chen et al., 1986). N-Acetylglutathione (NAcGSH) was prepared as follows. A solution of 0.3 g (0.49 mmol) of glutathione disulfide in 5 mL of H 2 0 was adjusted to pH 9.5 with N H 4 0 H . After addition of 1.5 equiv of acetic anhydride the solution was strirred at room temperature for 4 h with addition 1.5-equiv aliquots of anhydride added at 0.5-h intervals. The pH was maintained at 9.5 by periodic addition of N H 4 0 H . The reaction was stopped by adjusting the pH to 7.0 with acetic acid and lyophilizing. The residue, which was dissolved in 4 mL of H 2 0 , was stirred with 1 equiv of dithiothreitol under N 2 for 1.O h. The reaction mixture was immediately applied to a 2.4 X 70 cm bed of DEAE-cellulose equilibrated with 0.1 M ammonium acetate (pH 7.0). The column was eluted with a 1-L linear gradient of 0.1-0.5 M ammonium acetate (pH 7.0). Chromatography and fraction collection were performed under N2. Fractions containing NAcGSH were lyophilized repeatedly from water to give 74 mg (0.27 "01) of NAcGSH as a white hygroscopic powder. The product was judged 198% pure by 'H N M R and contained no detectable (
