MARCH/APRIL 1991 VOLUME 4, NUMBER 2 @Copyright 1991 by the American Chemical Society
Invited Review Glutathione S-Transferases: Reaction Mechanism, Structure, and Function Richard N. Armstrong Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742 Received October 4, 1990
I ntroductlon
chart I
The glutathione S-transferases (EC 2.5.1.18) catalyze the nucleophilic addition of the tripeptide glutathione (GSH; see Chart I) to substrates that have electrophilic functional groups. The enzyme is found in most aerobic microorganisms, plants, and animals. The primary function of the enzyme, particularly in higher organisms, is generally considered to be the detoxication of both endogenous and xenobiotic alkylating agents such as epoxides, a,D-unsaturated aldehydes and ketones, alkyl and aryl halides, and others. It is probably fair to say that this family of proteins is the single most important group of enzymes involved in the metabolism of electrophilic compounds. Certainly one of the most fascinating aspects of the biochemistry of the GSH transferases is their ability to catalyze reactions toward such a large number of structurally diverse substrates, a characteristic shared with most other detoxication enzymes. This ability is a consequence of the existence of several isoenzymes each of which has a unique substrate selectivity and the remarkable tolerance of each isoenzyme for both the type of electrophilic functional group and the structure of the molecule to which it is appended. I t is clear that a structural and mechanistic understanding of the substrate preferences of the GSH transferases is essential for the elucidation of their influence on the toxicology of alkylating agents. Although the GSH transferases occur in a large number of organisms, most of the mechanistic and structural work has been carried out on enzymes from higher organisms such as rats, mice, and humans. The bulk of the GSH transferase activity in these species is due to a collection of cytosolic isoenzymes all of which are dimeric proteins
with subunit molecular weights of about 25K. In the last 15 years considerable attention has been given to the classification of the large number of isoenzymes from various species (1-3). The early classifications, based primarily on substrate specificity and subunit molecular weight, have given way more recently to the development of a genetic catalogue of the proteins. The advances being made in the genetic classification of the isoenzymes and
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0 1991 American Chemical Society
Armstrong
132 Chem. Res. Toxicol., Vol. 4,No.2, 1991
the regulation of gene expression are of tremendous importance in unraveling the role of the GSH transferases in toxicology. However, they are not the subject of this review. These particular topics have been the subject of recent reviews ( 4 , 5 ) . A very brief discussion of the classification of GSH transferases is, however, warranted by way of introduction. In higher organisms there is very clear evidence for a t least three gene classes of the cytosolic enzymes. They have been designated a,p, and T . Each gene class consists of two or more genes that encode different subunit types. For example, the polypeptides encoded by a class genes in rat, designated subunits 1,2,8, and 10, combine to form active homodimeric or heterodimeric holoenzymes 1-1,2-2, 1-2, etc. p class genes encode subunit types 3,4,6,9, and 11 which similarly combine into homodimeric and heterodimeric holoenzymes. Inter-gene-class heterodimers are not known, a fact that suggests the interfacial regions for subunit interactions are unique, in this respect, for each gene class. Primary sequence homologies for subunits within a gene class are quite high, typically 270% sequence identity. Not surprisingly, homologies between subunits of different gene classes are significant but considerably less, usually about 40%. In addition to the cytosolic isoenzymes, there is a microsomal enzyme that bears little resemblance to any of the former except that it catalyzes the same reaction (6, 7). The coverage of this review is limited to the cytosolic enzymes. Although the actual reaction catalyzed by the GSH transferases is quite straightforward, relatively little is known concerning the details of how the enzyme actually accelerates the addition of GSH to the electrophilic substrate. The general reaction that is catalyzed is illustrated in eq 1. The central features of the chemical and kinetic E
+ GSH * EoGS- + H+
-
E.GS-*R-X E-GSR-X- + E + GSR
+ X-
(1)
mechanisms are that the nucleophilic species in the active site of the enzyme is the thiolate anion of GSH and that the reaction is sequential; that is, the addition reaction takes place in the ternary complex of enzyme, GSH, and electrophile. Over the last decade a considerable amount of information has been gathered concerning the kinetic mechanisms and substrate specificities of a number of different isoenzymes. Most of the detailed kinetic investigations of the enzyme-catalyzed reactions suggest that the addition of substrates is random. Equation 1 is misleading in this regard. However, given that the typical dissociation constant for GSH and the enzyme (10-30 pM) is 2-3 orders of magnitude lower than the normal concentration of GSH (1-5 mM) in healthy cells, the kinetic mechanism of the enzyme under physiological conditions is, for all practical purposes, ordered with GSH adding first. Much of the work on the kinetic mechanism and the more descriptive investigations of substrate specificity have been summarized in recent reviews ( 4 , 5 , 8 ) and will not be reiterated here in any detail. To fully appreciate the participation of the GSH transferases in the metabolism of xenobiotics, it is necessary to understand the chemical mechanism of catalysis as well as the influence of protein structure on substrate selectivity. It is the progress that has been made, particularly during the last three or four years, toward an understanding of these general issues that is the topic of this review. I t is convenient, if not entirely logical, to segregate the discussion of the chemical mechanism of catalysis into the issues that are somewhat more specific. First to be considered is the interaction of the enzyme with
s- s+ GS--H....B
- +
HB Figure 1. Two possible scenarios for the removal of the proton from the thiol of enzyme-bound GSH in catalysis. Polarization of the S-H bond by a general base (B)in the active site is illustrated on the left. Alternatively, the bound peptide may exist as the thiolate anion at neutral pH in the binary complex if the thiol is positioned in a positively charged electrostatic field represented on the right as BH'. GS
the physiological substrate GSH and the influence of the protein on the chemical properties of the tripeptide. Second, the experimental evidence for the putative involvement of particular amino acid residues in binding and catalysis will be evaluated. The third topic to be discussed is the nature of transition states and intermediates on the reaction coordinates of specific substrates. This latter point is intimately related to differences in the substrate selectivity observed with different isoenzymes. Finally, the current approaches to the elucidation of structurefunction relationships of the various isoenzymes and the design of catalysts with altered catalytic properties will be discussed.
Thiolate Ion and Catalysis It is generally accepted that the addition of thiols to electrophiles occurs by way of the thiolate anion. In fact, a recent estimate of the relative reactivities of simple thiols and thiolates in aqueous solution suggests that the thiolate anion is up to lo9 times more reactive than its conjugate acid (9). It therefore stands to reason that one role of the enzyme is to remove the proton from the thiol of GSH and generate the more reactive thiolate, GS-. Although there is no doubt that the proton must be removed at some point on the reaction coordinate, there are essentially two views as to how this might occur (10,ll). The two views differ with respect to the nature of the nucleophilic species in the active site. The species are essentially distinguished by the position of the proton in the transition state for the reaction as illustrated in Figure 1. The nucleophile in the general-base-catalyzedreaction shown on the left is a thiol with the S-H bond polarized by hydrogen bonding to a base in the active site. The alternative is preionization of the thiol to the thiolate assisted by a positively charged electrostatic field in the active site as shown on the right in Figure 1. The properties of the ground-state binary complex of the enzyme and GSH can help to differentiate these possibilities. That is to say, evidence for the existence of a significant fraction of the enzyme-bound peptide in the thiolate ionization state would be a strong indication for the preionization scenario. Several lines of evidence now suggest that, in fact, the predominate ionization state of the thiol of GSH in the binary complex is the thiolate (Le., EaGS-). Thiolate anions have a relatively strong absorption band in the ultraviolet at approximately 235 nm (e = 400043000 M-' cm-') which is attributable to an n o* electronic transition of a lone pair on the sulfur. A similar transition for the protonated thiol occurs at higher energy (195 nm). In spite of the considerable backbone absorption of the protein in the 235-240-nm region, it is possible to observe the thiolate anion of bound GSH by UV difference spectroscopy. Difference spectroscopy of the binary complex of isoenzyme 4-4 of rat liver GSH transferase with GSH and the enzyme alone or as the binary complex with the oxygen analogue (y-L-glutamyl-L-serylglycine) of GSH at neutral pH has revealed an absorption band at 239 nm (t = 5200 M-l cm-l) that is most reasonably assigned to E-GS-, the thiolate anion of the bound tripeptide (11). Furthermore,
-
Chem. Res. Toxicol., Vol. 4, No. 2, 1991 133
Invited Review
-
__
-
__
IpH=12pM
EH'
+ GSH L
pK,GSH= 9.0 EH'
+
\ GS-
KdGs
EH+GSH L pK,EGSH = 6.6 \
EH+GS'
-= 0.09 PM
Figure 2. Proposed binding and acid-base equilibria for GSH and isoenzyme 4-4. The dissociation constant for the thiolate anion from the enzyme is calculated as described in the text from the known pK,s of the enzyme-bound and free GSH and the observed dissociation constant of GSH measured at low pH.
E
t
B
A EH'
+ GO?-
T AGO&= AGk
+ AGD
- 1
\
AG,k
5
AGh
Figure 3. (A) Utilization of binding energy AGbe for the deprotonation of GSH in the active site of the enzyme. The actual observed binding energy for the protonated peptide AC,be is smaller than A G b by the energy required to destabilize the thiol, AGD (B)Realization of ACDas observed binding energy for the resonance-stabilizedcarboxylate analogues of GSH. The amount
of additional binding energy 6AGob actually realized with the carbox late analogues can be calculated from 6AGOh = -RT In ( & G S ~ K d U l d=~-2.4 e ) kcal/mol for 1.
titration of the difference absorption bands over the pH range of 5-8 indicates that the pK, of the thiol of bound GSH is in the range 6.4-6.7. This is about 2.5 pK units lower than the acid dissociation constant of GSH in aqueous solution (pKaGSH= 9.0) and is quite close to the pK, of 6.6 estimated for E-GSH from the pH dependence in the enzyme-catalyzed addition of GSH of kc/KmCDNB to l-chloro-2,4-dinitrobenzene(CDNB)under conditions of saturating GSH (12). The evidence cited above suggests that one role of the enzyme is to lower the pK, of the bound thiol so that a significant fraction is ionized at physiological pH. Given the pK, of both the enzyme-bound and free thiol and the dissociation constant (KdGSH) of the enzyme and GSH at low pH, it is possible to complete the thermodynamic box shown in Figure 2 by calculation of the dissociation constant for the thiolate; &''- = Kd'sH(K,GSH/KaE*GSH) =9 X M. The analysis reveals that the thiolate is bound much more tightly than is the conjugate acid. The enzyme can be viewed as utilizing some of the binding energy of the peptide to destabilize the thiol as is illustrated in Figure 3A. The fraction of the binding energy of GSH utilized to destabilize the bound thiol is given by AGD = RT In (KaE*GsH/K,GSH) = 3.3 kcal/mol. This analysis predicts that a good structural mimic of GS-, that is, a peptide that has a stabilized anionic group replacing the side chain of cysteine, should bind to the active site with much higher affinity than does GSH since some of the binding energy normally used to destabilize the thiol should be translated into observed binding energy. This particular situation is illustrated in Figure 3B. Two such molecules have been prepared, and their properties illustrate this point quite convincingly (11). Replacement of the cysteine side chain with a carboxylate group as in y-L-glutamyl-(D,L-%aminomalonyl)glycine(1) results i, I a mixture of diastereomeric peptides at least one
of which binds to the enzyme much more tightly (Kd = 7 X M a t pH 6.5)than does GSH (Kd = 2.2 X lo-' M). The facile epimerization about the a-carbon of the aminomalonyl residue precludes a direct determination of the dissociation constant for the correctly configured isomer. However, if it is assumed that only the L-configured epimer is responsible for the inhibition, then the carboxylate analogue binds about 60-fold more tightly than does GSH. This corresponds to a realization of an additional -2.4 kcal/mol of binding energy (6AGob) as observed binding energy in the carboxylate-containingpeptide. That 6AGob # AGD is simply a manifestation of the fact that the carboxylate analogue is not a perfect mimic of the thiolate. Just about the same result is obtained with the configurationally stable carboxylate analogue -p~-Glu-~-AspGly (2), which binds with a Kd of 9 X lo-' M. The incorrectly configured analogue -p~-Gl~-~-AspGly (3) binds somewhat M) than does GSH. less tightly (Kd = 4.7 X The rather pronounced affinity of the enzyme for the side-chain anion is an obvious indication of a positively charged electrostatic field in the active site of the enzyme, the function of which is to destabilize or lower the pK, of the thiol. The rest of the complementary surfaces of the enzyme and peptide presumably function to position the thiol in that electrostatic field. In the absence of a crystal structure of the enzyme several laboratories have resorted to what might be called "site-directed substrate mutagenesis", the alteration of the peptide substrate structure to probe enzyme-substrate interactions. A fairly large number of substrate analogues of GSH have been synthesized for this purpose (12-17),and a good deal of valuable information has been catalogued concerning the likes and dislikes of various isoenzymes with respect to specific modifications of the peptide structure (14-17). In some instances it is obvious that alteration of the peptide structure changes the relative orientation of the peptide and electrophilic substrates in the active site. For example, the stereoselectivity of the addition of the retro-inverso isomer of GSH (13) to phenanthrene 9,lO-oxide catalyzed by isoenzyme 4-4is quite distinct from that shown with the natural substrate (12). Although conjecture about the specific orientation of peptide and protein functional groups from these types of studies is entertaining, it is also potentially misleading without some notion of the mechanistic consequences or a knowledge of the protein structure. Unfortunately, only a couple of the alternative substrates have been pursued in any mechanistic detail (12), and virtually nothing is known about the protein structure at this time. Two alternative substrates, N-acetylGSH and Y-L-G~UL-CYS,which are reasonably good substrates for isoenzyme 4-4,have been shown, in one very important respect, to behave similarly to GSH in catalysis (12). The enzyme is capable of significantly lowering the apparent pK, of the thiol group of both of these alternative substrates when bound in the active site. Kinetically determined pK, values, which obviously should be viewed with considerable caution, suggest that the enzyme reduces the pK, of the thiol by as much as 2 pK units in the ternary complex of enzyme, analogue, and CDNB. But there is a difference; it is that the limiting values of k , for the two analogues at high pH are smaller by an order of magnitude than that for GSH. The apparent pK, of enzyme-bound NacetylGSH and Y-L-G~u-L-CYS (7.5and 7.7,respectively) are somewhat higher than that for E-GSH. What is unusual is that the nucleophiles (E-analogues)with the higher pK, are less reactive at high pH, where they are completely deprotonated, than is the less basic nucleophile, E-GS-.
134 Chem. Res. Toxicol., Vol. 4 , No. 2, 1991
This is contrary to the expected Bransted behavior where the nucleophiles of higher pK, should be at least as reactive as GSH (Bransted fin,, = 0) or more reactive (fin,, > 0). It is certainly possible, even likely, that the kinetically determined pK,s are not precise. However, another interesting possibility is that the analogues are not optimally oriented in the active site such that the thiolate is coordinated to another functional group on the protein or is more heavily solvated than it is in E-GS-. Both scenarios would have the effect of reducing the nucleophilicity of .the thiolate analogues, though to what extent is difficult to predict. Thus another role of the active site of the enzyme may be to shield the thiolate anion from solvent and enhance its reactivity. Other considerations suggest that desolvation of the nucleophile may be a key feature in catalysis. As suggested above, lowering the pK, of the thiol in the active site will, depending on the sensitivity of the transition state, actually decrease to a greater or lesser extent the effective nucleophilicity of the anion. The fact that transition states for addition of thiolate anions to electrophiles are relatively insensitive to the basicity of the nucleophile (fin,, typically < 0.5) suggests that a decrease in the pK, will only marginally decrease the reactivity of the anion and will be more than offset by the increased fraction of the anion (11,12, 18). Another way in which the enzyme might enhance the reactivity of the thiolate is to shield it from solvent. A recent investigation by Huskey et al. (19) has directly addressed this problem through the use of kinetic solvent isotope effects on both the specific-base- (spontaneous) and enzyme-catalyzed additions of GSH to CDNB. The inverse kinetic solvent isotope effect of 0.84 on the spontaneous reaction was used to estimate that the extent of desolvation in the transition state is a modest 3470, an observation that is consistent with the low sensitivity (&, = 0.16) of the reaction to the basicity of the nucleophile (12). Fractionation factors estimated for EsGS-, and the ternary complex EeGS-CDNB from the kinetic solvent isotope effects of near unity for k , and k,/KmGSHand of 0.79 for k,/KmcDm in the reactions catalyzed by isoenzyme 1-1suggest that a change in solvation of the thiolate ion occurs in proceeding from E-GS- to the transition state, a change similar in nature to that observed in the spontaneous reaction (19). More importantly, it appears that, in the enzyme-catalyzed reaction, the desolvation in the transition state is almost complete as compared to the partial desolvation apparent in the specific-base-catalyzed reaction.
Identification of Essential Catalytic Residues One of the first questions posed by mechanistic enzymologists in attempting to describe the catalytic mechanism of an enzyme is the following: What are the identities of the amino acid side chains that are essential for catalysis? For instance, apropos to the discussion above is the identity of the contributors to the electrostatic field that lowers the pK, of enzyme-bound GSH. There has been speculation that an imidazolium side chain of a histidine residue in the active site might be partially responsible for the electrostatic field that lowers the pK, of GSH ( 4 , 1 2 ) . This would be an intermolecular example of the type of thiol activation seen in the active site of papain, for example. There are a few reports that chemical modification of specific residues which might be involved in the deprotonation of GSH, namely, histidine (20) and arginine (21),leads to substantial inactivation of particular GSH transferases. Some investigators have interpreted this to mean that these residues are "essential" for catalysis. If
Armstrong the history of chemical modification of proteins and the more recent lessons of site-specific mutagenesis have taught us anything, it is that such conclusions should not be so quickly drawn. Van Bladeren and co-workers have presented evidence that modification of a cysteine residue at or near the active site by tetrachloro-1,4-benzoquinone and related compounds leads to irreversible inactivation of the p class isoenzymes (22, 23). These workers have suggested that even though cysteine is probably not an essential catalytic residue, there is a t least one side chain near enough to the active site to be a target for active site directed reagents. The availability of vectors for the heterologous expression of the mammalian enzymes in Escherichia coli (24-29) has encouraged the more the recent application of sitespecific mutagenesis to this problem. Inasmuch as most of this work has yet to appear in the primary literature, only a few highlights will be summarized here. Board, Mannervik, and co-workers (30) have prepared and characterized to some degree over a dozen site-specific mutants of the human isoenzyme e. The residues that were chosen as targets were among those found to be conserved among the various isoenzymes and for which some catalytic role might be imagined. For example, two highly conserved arginine residues appear to contribute to the binding of GSH since both the R13A and R20A mutants show diminished affinity for GSH and S-hexylGSH. It is possible that arginine side chains help to position GSH in the active site or constitute part of the electrostatic field that destabilizes the thiol of bound GSH. Further work on the mechanistic consequences of these and other mutations should be enlightening. The case for involvement of a histidine residue as a general feature in catalysis by the GSH transferases is not good. In the first place there is no highly conserved histidine in the sequences of the mammalian enzymes when all the gene classes are compared. There are, however, conserved histidines within a gene class. For example histidine residues 14 and 83 are conserved in the ~1 class isoenzymes, so it is conceivable that one or both might be crucial to the catalytic mechanism. Zhang et al.' have mutated each of the four histidine residues to asparagine in isoenzyme 3-3 from rat. None of the four mutants, H14N, H83N, H84N, and H167N, show any particular gross deficiency in catalyzing the addition of GSH to CDNB, phenanthrene 9,10-oxide, or 4-phenyl-3-buten-2one. In addition, 'H and 13C NMR studies' of the imidazole side chains of isoenzyme 3-3 show absolutely no perturbation in their pK,s upon binding of GSH, an observation that makes it highly unlikely that histidine side chains are involved in catalysis (31). Histidine is not an essential residues for isoenzyme 3-3, and it can be safely said that it does not play an immutable role in GSH transferase catalysis in general though it may participate in some ancillary fashion in other isoenzymes. To further emphasize the point, Lu and co-workers2have also found that the H8V, H143V, and H159V mutants of the (Y class isoenzyme 1-1from rat are quite active in the CDNB assay. Unpublished evidence3 also suggests that the cysteine residues in the p class isoenzymes, which are probably the targets for the chemical modification by tetrachloro-1,4benzoquinones,are not essential for catalytic activity. The single cysteine mutants C86S, C114S, and C173S and the double mutant N198K/C199S of isoenzyme 3-3, all of
'
P. Zhang, G.F. Graminski, and R. N. Armstrong, unpublishod results. A. Y. H. Lu, private communication. P. Zhang, and R. N. Armstrong, unpublished results.
Chem. Res. Toricol., Vol. 4 , No. 2, 1991 135
Invited Reuiew
Figure 4. Reaction coordinate diagram for the nucleophilic addition of GS- to l-chloro-2,4-dinitrobenzene. Catalysis by the enzyme beyond simple deprotonation the thiol must involve stabilization of the transition state for formation of the a-complex, the slow step. Such stabilization of the transition state is likely also to be apparent in stabilization of the intermediate iiself, particularly if it resembles the transition state.
which were made for crystallographic purposes, are good catalysts of the CDNB reaction. The inhibition observed after chemical modification is probably due to the introduction of a bulky group that either fills or perturbs the structure of the active site.
Chemical Mechanism and Substrate Specificity Nucleophilic Aromatic Substitution. The substrate specificity of any enzyme is a function of how effectively the enzyme can lower the activation barrier(s) for a particular chemical process. To truly understand the diversity in the catalytic specificity of the GSH transferase, it is essential to come to grips with the nature of the intermediates and transition states on the reaction coordinate. These can vary widely with the type of electrophilic functional group as well as the general topology of the substrate. Some progress has been made in sorting out the process of nucleophilic aromatic substitution reactions catalyzed by the enzyme. This reaction is illustrated in Figure 4 for the most common spectrophotometric substrate for the In solution, GSH transferases, l-chloro-2,4-dinitrobenzene. this general class of reaction is known to follow an addition-elimination sequence with the formation of a-complex or Meisenheimer complex intermediates. There is strong evidence with many nucleophiles and leaving groups that the rate-limiting step in the spontaneous reaction is the formation of the intermediate. If the rate acceleration provided by the enzyme involves something other than ionization and desolvation of the nucleophile, then the best place to look is at the transition state for a-complex formation. Does the enzyme, as illustrated in Figure 4,stabilize the transition state for formation of the a-complex? Several lines of evidence suggest that isoenzymes 3-3 and 4-4can do just that. The most commonly cited evidence for rate-limiting formation of the a-complex in nucleophilic aromatic substitution reactions is the increased reactivity of the substrate upon substitution of F- for C1-. The more electronegative leaving group is anticipated to facilitate formation of the intermediate but to have just the opposite effect on its decomposition. The second-order rate constant for the spontaneous reaction of GSH with l-chloro-2-nitro-4(trifluoromethy1)benzene is about 50-fold smaller than that for the 1-fluor0 compound. The kinetic constants k, and k , / K m for these two substrates with isoenzyme 4-4 differ by factors of 40 and 10, respectively, suggesting that the enzyme-catalyzed reaction has the same rate-limiting step as the base-catalyzed reaction (12). A similar observation has been made by Blanchard and co-workers with isoenzyme 6-6.* The differences between the kinetic con-
NO2
NO2
Figure 5. Reaction coordinate diagram for the equilibrium formation of the dead-end a-complex between GS- and 1,3,5trinitrobenzene. The enzyme stabilizes the a-complex by a factor of about lo3 and may also lower the barrier to its formation as indicated by the dashed line.
stants for the same two substrates in reactions catalyzed by isoenzyme 3-3are more modest, with the fluoro substrate being better by factor of only 4 (32). The lower sensitivity of isoenzyme 3-3 to the identity of the leaving group is perhaps significant since this isoenzyme is better than isoenzyme 4-4 at catalyzing nucleophilic aromatic substitutions. The sensitivity of the transition state in enzyme-catalyzed aromatic substitution reactions to electronic effects of the para substituent is instructive particularly when compared to the specific-base-catalyzed reaction (12). A Hammett plot of log k, vs d for a series of 4-substituted 1-chloro-2-nitrobenzenesubstrates and isoenzyme 4- giues a p value of 1.2, which is substantially smaller than the value of 3.4 found in the specific-base-catalyzed reaction. This finding is consistent with rate-limiting formation of the a-complex and perhaps an earlier or much later transition state for the enzyme-catalyzed reaction (12). A smaller substituent effect in the enzyme-catalyzed reaction is certainly compatible with the view that the thiolate in the active site is more reactive, thus requiring less electronic stabilization of the transition state. It is interesting that the substituent effect in the enzyme-catalyzed reactions with the two peptide analogues N-acetylGSH ( p = 1.9) and ~-L-G~u-L-CYS (p = 2.4) is greater than that for GSH. This observation is perhaps a reflection of a greater degree of solvation in the transition states for the two less reactive analogues. Direct evidence for the enzyme-catalyzed formation of the a-complex has been obtained from an investigation of the interaction of 1,3,5-trinitrobenzene with binary complexes of GSH with isoenzymes 3-3 and 4-4of the GSH transferases from rat liver (31,32). This electron-deficient arene, which does not posses a good leaving group, favors reversible accumulation of the a-complex in the active site of the enzyme at relatively low substrate concentration as illustrated in Figure 5. Isoenzyme 3-3 appears to have a particular capacity to stabilize the a-complex between GSH and trinitrobenzene (TNB). A comparison of the equilibrium constant, K,, for formation [GS-,,,, + TNB * MI of the Meisenheimer complex, M, to that of the enzymehound dead-end intermediate (EmGS- TNB * EeM) can tw made from the pH independent formation constants. The two lormation constants of 28 M-* for M and 9.6 X lo4 M I for E.M suggest that the enzyme stabilizes the in1,ermediateby as much as 4.8 kcal/mol. It is not known whether the much more favorable equilibrium in the active Mite is due to a larger rate constant for the forward reaction o r a smaller rate constant for decomposition of E.M.
+
K. K. Worig, G , Banks, and J. S. Blanchard, private communication.
Armstrong
136 Chem. Res. Toxicol., Vol. 4, No. 2, 1991 Scheme I 0
v v II
N02-
X
#&'y
II
NO1
Figure 6. Two posaible transition states for the enzyme-catalyzed addition of GSH to CDNB derived from BEBOVIB calculations and the observed inverse % and '4c primary kinetic isotope effects on the reaction. A late transition state for a-complex formation with unit C-S and C-Cl bond orders is shown on the left. Angles of the incoming nucleophile and the leaving group with respect
to the plane of the cyclohexadienate ring are indicated. The alternative of a late (product-like)transition state for decomposition (of the intermediate with complete C-S bond formation and substantial C-Cl bond cleavage is shown on the right. The figure is courtesy of K. K. Wong, G. Banks, and J. S. Blanchard!
Preliminary studies (33) of the preequilibrium kinetics of formation suggest that the enzyme does accelerate the forward reaction with a kl which is much larger than kl = 2.9 X lo3 M-ls-l (34) observed in aqueous solution. It therefore seems that the enzyme can also stabilize the transition state for formation of a a-complex and that the intermediate must resemble, to a first approximation, the structure of the transition state. The ability of a particular isoenzyme to stabilize M should be related to its catalytic efficiency toward nucleophilic aromatic substitution reactions if the analysis above is correct. Comparison of Kf(a a t pH 7.5 reveals that isoenzyme 3-3 stabilizes the l-($glutathionyl)-2,4,6trinitrocyclohexadienate anion about 70-fold more effectively than does isoenzyme 4-4. In most instances isoenzyme 3-3 is roughly an order of magnitude more efficient (judged by k,/K,) at aromatic substitutions than isoenzyme 4-4 so that the relationship appears to hold. Trinitrobenzene is, as expected, a very potent reversible inhibitor of the enzyme since the dead-end intermediate has a quite high affinity for the active site (32, 35). Evidence that the transition state for the isoenzyme 6-6 catalyzed addition of GSH to CDNB resembles the structure of the a-complex intermediate has been obtained through other means by Blanchard and co-~orkers.~ Both %S (%S-GSH)and I4C (14C-CDNB)primary kinetic isotope effects on k,/Km for this reaction are inverse (35S,kJK, = 0.971 f 0.005; 14C,k,/K, = 0.964f 0.010).These values, when matched to the isotope effects predicted for various transition-state structures modeled by BEBOVIB calculations, suggest two possible alternatives for the transition-state structure as shown in Figure 6. The isotope effects are compatible with a transition-state structure that resembles either the structure of the intermediate (a late transition state for a-complex formation) or the structure of the product (a late transition state for decomposition of the u-complex). Wong, Banks, and Blanchard4 have concluded that the first possibility is more likely given the ,preponderance of evidence that formation of the intermediate is rate-limiting. The dead-end intermediate complex formed with trinitrobenzene is also a useful tool with which to study the ionization behavior of GSH and perhaps other functional groups in the active site of the enzyme. The pH dependence of complex formation on the enzyme is not simple. The dependence of log Kf on pH clearly suggests that the equilibrium with isoenzyme 3-3 is affected by multiple ionizations (32). Our original analysis of the pH dependence of Kf concluded that a-complex formation occurred in a simple collision complex between TNB and E-GS-, was
isoenzyme 4-4
X
+ XA /
Ad A
(4R)
B (4s)
dependent on two ionizationsin the active site, and ignored the rather curious sigmoidal depecdence of the log c on pH. In this analysis the pKa of enzyme-bound GSH was estimated to be 5.7 with an additional ionization of another group with pK, of 7.6. Cleland subsequently derived a model that incorporates the dependence of c (maximum color formation) on pH assuming the formation of a colorless collision complex followed by a unimolecular step for formation of the a-complex and a pH dependence in the internal equilibrium between the colorless complex and the complex.^ A t high pH the internal equilibrium constant is very large, but at low pH it is assumed to be 0.61, the factor by which c changes going from high to low pH. Cleland's model predicts a pKaEeGSH = 6.7, which is in good agreement the with the pK, measured by other techniques with isoenzyme 4-4 (9, IO). Preequilibrium kinetic studies which should help to sort out this intriguing system are in progress. Michael Additions and Epoxide Ring Openings. The evolutionary development of catalytic specificity in the GSH transferases is an interesting matter on which to speculate. Different electrophilic functional groups do have different requirements with respect to catalysis. However, the reactions involving the addition of GSH to a,p-unsaturated aldehydes and ketones and epoxides appear wholly unrelated to nucleophilic aromatic substitution, yet all share a common need for stabilization of anionic intermediates or transition states. The evolutionary pressure for the detoxication of Michael acceptors and epoxides can be readily imagined. So, have specific isoenzymes evolved to accommodate particular functional groups and transition states? Or does each isoenzyme just posses some general but slightly modified ability to stabilize anionic transition states? Mechanistic investigations can perhaps help to define such questions if not answer them directly. The Michael addition of GSH to 4-phenyl-3-buten-2-one (Scheme I, X = H) catalyzed by isoenzyme 4-4from rat is stereoselective such that a 9:l ratio of the two possible diastereomeric products is observed (36). Clearly, the transition state for addition of GSH to one prochiral face of the enone is preferred. The question arises as t,o whether this stereochemical preference is mechanism based (i.e., stereoelectronic in nature) or is simply a consequence of some steric interference with one possible mode of binding. Were the latter the case, one might, anticipate essentially the same stereoselectivityof the enzyme toward a series of sterically similar substrates regardless of their reactivity. Just the opposite has been found for R series ~
~~
W. W. Cleland, private communicaGin The alternative nitdel derived by Cleland gives a considerably better stetistical annlyRiR than d t ~ our original calculations.
Chem. Res. Toxicol., Vol. 4, No. 2, 1991 137
Invited Review
Table 1. X-ray Diffraction Quality Single Crystals of GSH Transferase species isoenzyme, gene family human, GST2, a
space group monoclinic C2
rat, 3-3, p
monoclinic C2
bovine, neutral, r
tetragonal,
human, acidic, r
P&2,2 or &2,2 tetragonal, P41212or P&2,2
unit cell dimensions,
A
a = 100.8, b = 95.4,
c = 105.2, p = 92.4' a = 88.4, b = 69.4, c = 81.3, 0 = 106.0' a = b = 61, c = 237 a = b = 60.1, c = 244
of para-substituted 4-phenyl-3-buten-2-onesin which the reactivity of the enone was varied by changing the electron-withdrawing ability of the para substituent. The degree of stereoselectivity actually decreases with increasing reactivity of the substrate in a manner that is consistent with differential electronic stabilization of the two possible transition states. The enzyme exhibits little stereoselectivity toward more reactive substrates (X = NOz,66% product A) and a much higher stereoselectivity toward the least reactive substrate (X = OCH,, 95% product A). The log of (kC/K,Job,for the addition of GSH to a series of para-substituted 4-phenyl-3-buten-2-onesfollows the linear free energy relationship given in eq 2 (36),where log
(kc/Km)obs
- CappT = Papp'
(2)
u is the electronic substituent constant, T is the Hansch hydrophobic substituent constant (37),and pappand caPp are measures of the sensitivity of the rate constant to the electronic and hydrophobic nature of the Substituent. Of course, as illustrated in Scheme I, (k,/K,,,),b is a composite rate constant that represents a weighted average for the reaction proceeding through the two possible parallel transition states and is dominated by the transition state of lowest activation energy. The apparent substituent effects pappand c, are therefore a linear combination of the substituent effects for each transition state A' and B'. Measurement of the stereoselectivity (product ratio) allows a measurement of the individual substituent constants such that P A = 0.78,CA = 0.44, pB = 1.74,and CB = 0.51. It can be readily seen that the sensitivity of the two transition states to the hydrophobicity of the substituent is roughly the same, CA = cB, but that there is a substantial difference in the electronic sensitivity, P A < PB. Thus the transition state for the reaction which is least effectively catalyzed by the enzyme is considerably more sensitive to the presence of an electron-withdrawing group. Furthermore, since the log of the ratio of the two products reflects the difference in free energies of activation for the two diastereomeric transition states, it can be readily shown that a linear free energy relationship exists between this quantity and u as expressed in
log ([Al/[BI) - (CA - CB)* =
(PA
-P
B ) ~
(3)
where p o b = P A - PB = -0.94 and cobs = CA - CB = -0.07. One possible interpretation of these results shown in Figure 7 suggests that one transition state, A', is much less sensitive to the nature of the substituent because the enzyme provides additional electrostatic stabilization of the enolate, perhaps through a specific functional group (BH+) in the active site. This stabilization is not available in transition state B' so that formation of product B is much more sensitive to the reactivity of the substrate itself. This type of mechanistic observation tends to support the notion that specific isoenzymes may have evolved to deal specifically and stereospecificallywith particular functional groups.
2.49
asymmetric unit two dimers
478.5
2.3
882 881
unit cell volume, nm3 1012
V,,
resolution,
A
ref
2.7
43
one dimer
c2.0
41
2.4
one dimer
2.6
42
2.45
one dimer
