Structure and function of glutathione S-transferases

BB.

ELSEVIER

Biochim~ic~a et BiophysicaA~ta

Biochimica et Biophysica Acta 1205 (1994) 1-18

Review

Structure and function of glutathione S-transferases Matthew C.J Wilce, Michael W. Parker

*

St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Vic. 3065, Australia (Received 27 April 1993; revised manuscript received 5 October 1993)

Key words: G l u t a t h i o n e S - t r a n s f e r a s e ; P r o t e i n s t r u c t u r e ; X - r a y c r y s t a l l o g r a p h y ; E n z y m e m e c h a n i s m

Contents 1. Introduction

..............................................................

1

2. Comparison of primary structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

3. Structural studies of pi-class GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

4. Structural studies of mu-class GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

5. Structural studies of alpha-class GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9

6. Structural studies of other GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11

7. Heterodimer formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11

8. Location of the non-substrate binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11

9. Conformational changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11

10. Chemical modification studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

11. Catalytic m e c h a n i s m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14

12. Comparison to other glutathione-binding enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14

13. Basis for substrate selectivity--the substrate binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15

14. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

15. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

16. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

1. Introduction * Corresponding author. Fax: + 61 3 4162676. Abbreviations: GST, glutathione S-transferase; GSH, glutathione; r.m.s., root-mean-square

0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 3 ) E 0 2 2 7 - S

Glutathione a family

S-transferases

of multi-functional

(GSTs) enzymes

(EC 2.5.1.18) are involved

in the

2

M.C.J. Wilce, M.W. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

cellular detoxification and excretion of many physiological and xenobiotic substances. They catalyse the nucleophilic addition of the thiol of reduced glutathione (y-glutamyl-cysteinyl-glycine) to electrophilic centres in organic compounds. The glutathione conjugates so-formed are rendered more water-soluble, thus facilitating their eventual elimination. This reaction is one of the early steps along the mercapturic acid pathway in which hydrophobic xenobiotics are inactivated and eliminated from the organism [1]. An ATPdependent efflux pump that mediates the export of glutathione conjugates from cells has recently been described [2,137]. Mammalian GSTs can be grouped into at least five evolutionary classes designated alpha, mu, pi, theta and microsomal based on substrate and inhibitor specificities, antibody cross-reactivity and primary structures [3,4]. Recently an enzyme involved in the synthesis of leukotrienes, human leukotriene C 4 synthase, has been identified as belonging to a new class of GSTs [138]. The cytosolic GSTs exist as either homo- and heterodimeric forms due to multiple genes and monomer hybridization. Heterodimers have not been identified between molecules of different classes. The often-observed hetereogeneity of GSTs may be further complicated by covalent post-translational modifications such as glycosylation [5]. Members within any class exhibit similar monomer sizes (about 24 to 28 kDa), share high amino-acid sequence identity (typically 60 to 80%) and have distinctive but overlapping substrate specificities. The human alpha-, mu- and pi-class GSTs are thought to be products of at least six gene loci: A1, A2 (alphaclass), M1, M2, M3, M4, M5 (mu-class) and P1 (pi-class) [6,139,140]. It is highly likely that GSTs evolved from a common gene, with the exception perhaps of the microsomal GST which shows no obvious sequence relationship with the cytosolic enzymes [7]. Cytosolic GSTs have been most extensively studied in human, rat and mouse tissues where they are most abundant. In human liver they comprise about 5% of the total cytosolic protein. The pi-class enzyme is the most widely distributed isoenzyme and usually the most abundant [8]. The expression of the different isoenzymes is highly tissue-specific. For example, the alphaclass enzyme is the major isoenzyme in the human liver and kidney, whereas the pi-class enzyme is predominant in placenta, erythrocytes, breast, lung and prostate [9,10]. GSTs appear to be fairly ubiquitous amongst aerobic organisms. They have also been purified from

plants [11], fish [12], insects [13,14], fungi [15,16], yeast [17] and more recently, bacteria [18-21]. One of the fascinating aspects of GST enzymology is their ability to catalyse reactions towards a large number of structurally diverse substrates. Examples include alkyl- and arylhalides, lactones, epoxides, quinones, esters and activated alkenes [22]. Although the range of substrates that GSTs recognize is broad, they do share the common feature of being mostly hydrophobic and bearing an electrophilic centre. Initially the nucleophilic attack of GSTs was thought to be directed towards electrophilic carbon atoms but it was later established that electrophilic nitrogen in nitrate esters, sulfur in organic thiocyanates and disulfides, and oxygen in organic hydroperoxides could serve as alternative targets. The specific activities of GSTs towards certain substrates has proved useful in classifying new GSTs. For example, alpha-class GSTs are highly reactive towards cumene hydroperoxide, mu-class GSTs have a preference for epoxides whilst pi-class GSTs display high reactivity towards ethacrynic acid. However, such classifications can be misleading as, for example, a rat alpha-class GST has been shown to exhibit high reactivity towards aflatoxin B1-8,9-epoxide [141]. The cytosolic enzymes have two active sites per dimer which behave independently of one another [23]. Each active site consists of at least two ligand binding regions; the GSH binding site is very specific for glutathione, whereas the binding site for the electrophilic substrate (the substrate binding site) is less specific in keeping with the ability of GSTs to react with a wide variety of toxic agents. Some GSTs have been shown to have secondary catalytic activities including a selenium-independent peroxidase activity with organic hydroperoxides [24] and steroid isomerization [25]. In addition to their catalytic activity, GSTs may function as intracellular transporters of various non-substrate hydrophobic compounds such as bilirubin, heme, steroids, thyroid hormones and bile salts [9]. This binding often results in inhibition of the enzyme's catalytic activity [9]. GSTs have been implicated in the development of the resistance of cells and organisms towards drugs, pesticides, herbicides and antibiotics [22,27,19]. In particular, the overexpression of GSTs in tumours appear to be a factor in the development of acquired resistance towards anti-cancer drugs and hence GSTs are a therapeutic target for rational drug design [28].

Fig. 1. Alignment of consensus GST amino-acid sequences. The alignments of intra-class sequences were performed using the PILEUP program in the GCG package (gap weight = 3.0, gap length weight = 0.10) [131]. Consensus sequences were calculated with PRETTY from the GCG package [131]. Sequences were extracted from the SWISSPROT database [132]. Invariant residues are shown boxed. The locations of alpha-helices (denoted a) and beta-strands (denoted /3) are indicated. Residues located in the GSH and substrate binding sites are marked by the letters G and H, respectively.

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M.C.J. Wilce, M.W.. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

A detailed understanding of the enzymatic mechanism and the basis for substrate discrimination requires a knowledge of the three-dimensional structure of each GST isoenzyme. There has been intense interest in obtaining three-dimensional structures of GSTs over the last few years [29-40] with a concomitant increase in the number of site-directed mutagenesis studies. The crystal structures of GSTs from three of the major classes have recently been determined [35,37,38,40] and thus it is timely to review the recent GST literature in light of these structures. For different perspec-

tives, the interested reader is directed towards a number of excellent reviews which cover the historical [9,26], biochemical [22,41], genetic [6,42,142] and cancer aspects [28,43,44] of the field.

2. Comparison of primary structures Alignments of consensus amino-acid sequences from the major GST classes are presented in Fig. 1. Unless otherwise stated, the residue numbering of the human

Table 1 Sequence homologies between GSTs

a

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m

1 P h

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a

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t h t a

c h i c k

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l J--

I ~

55 55 55 55 55 54 84 84 84 84 84 48 48 48 47

* 9 9 9 9 6 5 5 5 5 5 5 5 5 5 * 9 9 9 6 5 5 5 5 5 5 5 5 5 * 9 9 6 5 5 5 5 5 5 5 5 5 * * 6 5 5 5 5 5 5 5 5 5 * 6 5 5 5 5 5 5 5 5 5 * 5 5 5 5 5 5 5 4 5 * 9 9 9 9 4 4 4 4 * 9 9 * 4 4 4 4 * 9 9 4 4 4 4 * 9 4 4 4 4 *4444

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1 e n s

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Sequences were extracted from the SWISSPROT database [132] and initially aligned using the PILEUP program of the GCG package [131]. The alignment was subsequently optimized with LINEUP [131]. Sequence homology was calculated using DISTANCES (match threshold 0.6) [131]. The homology values take into account conservative substitutions and thus are more sensitive indicators of structural relatedness. Each number in the table refers to the pairwise percentage sequence homology, divided by ten and rounded to the nearest integer. * denotes sequence identities between 95 and 100%. Only values above 35% are shown. Sequence legend: pi: pi-class GSTs from human, bovine, pig, mouse, rat and nematode; mu: mu-class GSTs from human, mouse, hamster, guinea pig and rat; alpha: human, rabbit, rat and mouse; theta: theta-class GSTs from rat; fish: plaice GST; lens: GST from octopus lens; fluke: fluke GSTs from fasciola and schistosoma; insect: drosophila, house fly, blowfly, mosquito; plant: maize, tobacco, silene, wheat, carnation; bacteria: Proteus mirabilis; dcma: dichloromethane dehalogenase; ssp: starvation stringent protein.

M. C.J. Wilce, M. W. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

placental pi-class enzyme will be used throughout the paper. The sequences vary in length from approx. 210 amino acids in the pi-class enzymes to 300 in the plant enzymes. SIX residues are conserved in all the known sequences of GSTs: they are Tyr-7, Pro-53, Asp-57, Ile-68, Gly-145 and Asp-152. Crystallographic studies have shown that the first two residues play key roles in the GSH binding site, whereas the role of the other residues is less clear. The last three residues are buried and may be important for determining the protein fold. The loss of free energy acquired in burying Asp-152 appears to be compensated by multiple interactions of the carboxylate moiety with side chain hydroxyl groups and main chain atoms. The consensus motif [S,T][R,N]AIL centred about residue 67 appears to be characteristic of GSTs although it is by no means unique to GST sequences. (Out of 31 808 entries in the latest releases of the SWISSPROT and PIR protein sequence data bases, 40 entries contain this motif but only 25 are GSTs). In many cases the mammalian classification of GSTs can be extended to other organisms on the basis of sequence comparisons. Pairwise sequence homologies (where conservative substitutions are taken into account) are presented in Table 1. On the basis of the table, there appear to be both alpha- and mu-class chicken sequences and three of the liver fluke GSTs can be assigned as mu-class sequences. Some of the insect, fish and bacterial sequences can be classified as Theta class sequences. It is important to stress that many of these classifications rely on low levels of sequence homology only and thus may be misleading. The assignments need to be tested further by detailed biochemical comparisons. A number of unexpected but significant sequence homologies with other proteins have been documented. These include some dehalogenases that catalyse the conjugation of glutathione to haloalkanes or chlorinated quinones in three species of bacteria [45-47,143]. Squid lens proteins have been identified as GSTs although they probably fulfil a purely structural role as lens crystallins [48,49]. Buetler and Eaton [144] have proposed that the lens GSTs be grouped into a new class termed Sigma [144]. Matches have been made to a bacterial stringent starvation protein [50] and to an ethylene-responsive flower senescence-related protein [51]. The functional significance of both matches is unclear and open to speculation. It has been argued that glutathione-dependent enzymes evolved in aerobic organisms in response to the generation of the toxic by-products of oxygen metabolism [for example, 52]. The conservation or conservative substitution of key residues throughout the length of all sequences suggest GSTs evolved by divergence from a common ancestral gene. The exon/intron boundaries observed in the mammalian alpha-, pi- and

5

mu-classes are highly similar, suggestive that the evolution and diversification of GSTs may be due in part to exon shuffling. The successful production of active chimaeric GST constructs supports the hypothesis [53,54]. It has been proposed that Theta-class GSTs may have been the evolutionary forerunner of the alpha-, mu- and pi-class enzymes based on the apparent distribution of the former in a diverse range of organisms [55,144]. The other GST classes would have arisen by duplication of the Theta gene allowing organisms to adapt to various toxic stresses during the course of evolution. Hydropathy plots and secondary structure predictions based on the primary sequences suggested that the cytosolic GSTs had similar chain folds consisting of alternating a-helices and /3-strands [56,57]. This pattern was consistent with the canonical TIM barrel fold first characterized in the structure of triosephosphate isomerase [58] and which has subsequently been identified in 15 other enzymes to date [59]. These predictions were subsequently proved wrong when the first threedimensional structure of a GST was determined in 1991 [35]. This emphasizes the caution that should be applied when judging the results of secondary structural predictive algorithms.

3. Structural studies of pi-class GSTs The first pi-class GST to be crystallized was from bovine placenta in 1988 [30]. Unfortunately, the crystallization conditions did not prove reproducible between protein batches and furthermore the diffractions patterns from one crystal to another were not entirely identical (O. Gallay, personal communication). Subsequently, the same group purified and crystallized a pi-class GST from pig lung [34]. The porcine enzyme crystallized in a different sjgace group and diffracted to a higher resolution (2.2 A) than the bovine enzyme, despite both enzymes possessing very similar isoelectric points and exhibiting more than 80% residue identity. It was these crystals that lead to the first three-dimensional structure determination of a GST in 1991 [35]. It is worth describing the structure of the pi-class enzyme in some detail here, as its polypeptide fold has turned out to be archetypical for the whole family of cytosolic GSTs. The polypeptide fold is composed of two domains as shown in Figs. 2A and 2B. The Nterminal domain (residues 1 to 76) consists of a central mixed /3-sheet of four strands (/31 to /34) flanked on one side by two a-helices ( a l and a3) and on the other side, facing the solvent, another helix (a2). The latter helix appears quite mobile as it exhibits elevated temperature factors (see Fig. 9). Helices a l and a3 lie packed against the /3-sheet with their axes running parallel to the strands. The C-terminal domain (re-

M. C.J. Wilce, M. W. Parker / Biochimica et Biophysica Acta 1205 (1994) 1-18

sidues 81 to 209) consists of a right-handed bundle of five a-helices (a4 to a8), the first two of which are longer than 40 A. These two helices pack in anti-parallel fashion and appear distinctly crescent-shaped. The bent appearance of a5 is probably due to the presence of two prolines (Pro-123 and Pro-128) in the helix. The C-terminal domain fold shares a topological relationship to that of annexin [60] although no functional conclusions can be drawn from this relationship. The interface between the domains is roughly V-shaped (Fig. 2A,B), with closely-packed residues participating in hydrophobic interactions at the base that are gradually replaced by salt links and hydrogen bonds as the domain surfaces separate. The two domains are connected by a short peptide segment, residues 77 to 82, with the sidechain of Tyr 79 wedged between them. The structure of the dimer is presented in Fig. 3A and B. The molecule is oglobulaor with approximate dimensions of 45 ,~ x 55 A × 60 A. The twofold relationship between subunits is almost exact with a rootmean-square (r.m.s.) deviation in alpha-carbon coordinates of 0.2 A. The dimer interface is fairly extensive with a total of about 1500 ~2 of surface area buried between monomers. This value is within the range normally found for dimers of similar subunit molecular weights [61]. The side-on view of the dimer, presented in Fig. 3B, shows the interface to be roughly V-shaped. From a well-packed base, the monomer interfaces diverge into the helical towers of the C-terminal domains. The interface is formed by a mixture of hydrophilic and hydrophobic interactions. Unfolding studies have demonstrated the importance of the dimer structure for activity [62,63]. In one case, structured monomers have been isolated as an unfolding intermediate but found to be devoid of activity [62]. The GSH binding site of the enzyme could be identified due to the presence of the competitive inhibitor, glutathione sulfonate in the crystals of the porcine enzyme. The site was located in a deep (15 ,~) V-shaped cleft formed by the abutting of the two domains, with domain 1 providing most of the contacts. The active site of each monomer is well-separated in the dimer with the closest atomic distance between glutathione molecules of about 14 ~, (Fig. 3A). The inhibitor bound in an extended conformation similar to that found in crystals of reduced glutathione [64] and in some other glutathione-binding enzymes [65,66]. The glutathione moiety of the inhibitor bound to the GSH binding site with its 3,-glutamyl arm pointing downward toward the dimer interface and the glycine pointing towards the

7

A

B

Fig. 3. (A), (B) Ribbon representations of the pi-class dimer. Residues were assigned to secondary structural elements according to criteria defined by Kabsch and Sander [133]. The S-hexylglutathione inhibitor is shown in ball-and-stick fashion. Two orthogonal views are shown. The figures were produced by the computer program MOLSCRIPT [134]. Helix a2 has been omitted in (B) for clarity.

surface of the N-terminal domain as shown in Fig. 3A. Details of the inhibitor interactions with the protein are shown in Fig. 4A. There are a total of 21 potential hydrogen-bonding interactions with the glutathione moiety, which explains the high stereospecificity of GSTs for glutathione. Of particular interest was the interaction between the glutathione sulfur and Tyr-7. This tyrosine was one of the very few residues conserved in all known GST sequences suggesting an im-

Fig. 2. Ribbon representations of GST crystal structures. Residues were assigned to secondary structural elements according to criteria defined by Kabsch and Sander [133]. The locations of alpha-helices (denoted a) and beta-strands (denoted /3) are indicated. Inhibitors are shown in ball-and-stick fashion. Two orthogonal views are shown. The figures were produced by the computer program MOLSCRIPT [134]. (A), (B) pi-class human GST [37] (C), (D) mu-class rat GST [38] (E), (F) alpha-class human GST [40].

8

M.C.J. Wilce, M. IV.. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18 ,~r

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the human placental pi-class enzyme in complex with the competitive inhibitor S-hexylglutathione [37]. The substrate binding site was located adjacent to the GSH binding site, with the hexyl moiety sitting on top of the loop connecting fl-strand 1 to a-helix 1, nestled in a hydrophobic cleft (see Fig. 3A). The cleft is coated by the side-chains of Tyr-7, Phe-8, Pro-9, Val-10, Val-35 and Tyr-106. The possible existence of other substrate binding sites adjacent to the GSH binding site cannot yet be ruled out. The structure of the human enzyme was found to be virtually identical to that of the porcine enzyme; not an unexpected result given the 82% sequence identity for the pair. Furthermore, the specific interactions between GSH binding site residues and glutathione were also identical, thus confirming the importance of the contacting residues identified in the porcine structure.

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131

Fig. 4. Schematic drawing of residues that interact with glutathione in the G S H binding site. Potential hydrogen bonds are indicated by dashed lines. (A) pi-class G S T [37] (B) mu-class G S T [38] (C) alpha-class (5ST [40].

portant role for the residue in the catalytic mechanism (Fig. 1). The consensus motif [S,T][R,N]AIL centred about residue 67 is located at the N-terminal end of helix a3 with the s e r i n e / t h r e o n i n e residue involved in glutathione-binding. The motif thus contributes residues to both the GSH binding site and to the domain interface. The symmetry-related subunit provides a salt bridge to glutathione binding, demonstrating the importance of the dimeric state for activity. The porcine structure did not unambiguously reveal the substrate binding site to which electrophilic substrates bind. The location was later established with the crystallization [33] and structure determination of

Crystals of mu-class GSTs have been reported from rat liver [29,33]. It is of interest to note that crystals grown in the presence of the weak inhibitors methyl- or ethylmercury chloride appear to be isomorphous with crystals grown with a glutathione-based inhibitor, whereas completely different crystals can be grown in the absence of inhibitor. Structural studies of these different crystal forms will indicate whether there are any significant comformational changes on ligation and, if so, will reveal the molecular details of any such changes. To date, only the structure of the mu-class enzyme in complex with the physiological substrate glutathione has been reported (Fig. 2C,D) [38]. Despite the low level of sequence identity (31%), the mu-class enzyme shares the same overall folding topology as the pi-class enzyme. Domain 1 spans residues 1 to 82 and Domain 2 spans residues 90 to 217 with a short linkage region connecting the two domains. A major difference between the two structures is the presence of a long loop between fl-strand 2 and a-helix 2 in the mu-class enzyme (residues 33 to 42); the so-called mu-loop (Fig. 2C). The mu-loop results in a deeper active site cleft than seen in the pi-class structures. The glutathione binds to the enzyme in an extended conformation as was found in the pi-class crystals. In total, there are twenty hydrogen bonds or salt bridges involved in binding glutathione to the enzyme as shown in Fig. 4B. As in the pi-class structures, a hydrogen-bonding interaction is observed between the thiol sulfur of glutathione and the hydroxyl of the conserved tyrosine. There are seven residues found contacting the glutathione in the pi-class enzymes that are conserved in the mu-class sequences and all seven fulfil the same contacting role in both enzymes.

M.C.J. Wilce, M.W. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

An exciting facet of the mu-class structural work has been the demonstration that the crystals are chemically competent to form a Meisenheimer or o--complex between glutathione and 1,3,5-trinitrobenzene [67]. The crystal structure of this complex might explain how the enzyme stabilizes intermediates and transition states of nucleophilic substitution reactions. The substrate binding site has not yet been identified in a mu-class GST by crystallography. However, the active site of the human muscle enzyme has been probed by nuclear magnetic resonance spectroscopy [36]. The substrate binding site was located by use of a spin-labelled probe conjugated to glutathione and implicated Tyr-ll6, Ala-38, Ala-ll2, Ala-213, Met-35, Met-105, Met-109 and Met-212 as being in or close to the substrate binding site. Modification studies with the glutathione analogue, S-(4-bromo-2,3-dioxobutyl) glutathione, support the conclusion that Tyr-ll6 is located in the substrate binding site [68]. A superposition of the Pi and Mu crystal structures shows the proposed substrate binding sites of the two are in equivalent positions. As mentioned earlier, evolution and diversification of GSTs may be due in part to exon shuffling. If this were the case, then one would expect that the exon/ intron boundaries would not be randomly distributed with respect to the protein fold. Indeed it is found that each exon encodes a discrete structural element with exon/intron boundaries positioned in loop regions as shown in Fig. 5 (Armstrong, R.N. and Gilliland, G.L., unpublished data).

Fig. 5. Correspondence of exons with the rat mu-class crystal structure. Structural elements encoded by consecutive exons are marked by alternate light and dark shading. E x o n / i n t r o n boundaries are derived from the rat YB z gene [135].

'

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Residue Number

Fig. 6. Superposition of GST crystal structures. The positional deviation of the alpha-carbon atoms is plotted as a function of residue number. The superposition was based on overlaying m o n o m e r s (solid line) and N-terminal domains (dashed line) using the algorithm of R o s s m a n n and Argos [136]. The overall r.m.s, deviations are also given.

5. Structural studies of alpha-class GSTs

Crystals of alpha-class GST have been reported by two groups. A human chimaeric GST has been crystallized in the presence of the weak inhibitor methylmercury chloride [33]. Crystals of human alpha-class GST (AI-1) were obtained in the presence of S-(2iodobenzyl)glutathione [31]. The crystals of the two GSTs appear isomorphous despite the fact they crystallize in the presence of two very different inhibitors. The structure of human GST AI-1 has just been determined [40]. The polypeptide fold of the alpha-class GST closely resembles the fold first observed in the structure of the pi-class enzyme. The main difference is due to the extra residues at the C-terminus of the alpha-class enzyme (Fig. 1). The C-terminus folds into an additional alpha helix (a9), positioned so that it blocks part of the substrate binding site end of the active site from solvent (Fig. 2E,F). The alpha-class enzyme does not have a large insertion of residues between /32 and a2 (Fig. 1) and thus does not have a mu-loop. The sequence identity of the alpha-class structure to the mu- and pi-class structures, based on three-dimensional structural alignment, is about 20% and 32% respectively [40]. Nevertheless, the r.m.s, alpha-carbon deviations on superposition of the crystal structures are quite close as shown in Figs. 6 and 7. The r.m.s, deviations are appreciably lowered when individual domains are superimposed, demonstrating the close similarities of the individual domain folds and

M. C.J. Wilce, M. W. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

10

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ations of g l u t a t h i o n e , S-

'th~oln~c derived from the crystal based on overlaying the coordidomains. The ligands are overof the human pi-class enzyme. The in stick fashion and the h y d r o p h o b i c

i

' _ ~k~'-~.~.~, . _ ~

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Fig. 7. O v e r l a y s o f s u p e r p o s e d a l p h a - c a r b o n c o o r d i n a t e s of G S T crystal structures. ( A ) pi-class vs. m u - c l a s s (B) pi-class vs. alpha-class (C) mu-class vs. alpha-class.

the presence of significant rigid body rotations of subunits and domains relative to one another. It remains to be seen whether the amount of rotation between subunitsand domains will turn out to be characteristic of a particular GST class. Domain 1 shows the greatest similarities with the major differences being the muloop insertion that is unique to the mu-class enzyme and the positional flexibility of helix a 2 relative to the beta-sheet of domain 1 seen in all three classes. Although glutathione binds to the same site in all the GST crystal structures, some of the detailed interactions are different. The polar interactions between glutathione and the alpha-class enzyme are shown in Fig. 4C. Again the hydroxyl of the conserved tyrosine (Tyr-9) is found in hydrogen-bonding distance of the glutathione sulfur atom. There appears to be a further interaction of the sulfur atom with Arg-15, an interaction not observed in the other classes. Interactions between conserved glutamine and aspartate residues with the y-glutamyl moiety are retained in all four crystal structures. The location of the substrate binding site has been defined by the position of the benzyl moiety of the inhibitor which sits in a similar but not identical position to that observed for the hexyl chain in the human pi-class enzyme (shown in Fig. 8). The fact that the binding is not identical is a consequence of the differing topologies of the substrate binding site between isoenzyme classes, which is discussed in more detail later.

M.C.J. Wilce, M.W. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

6. Structural studies of other GSTs So far, structural studies have tended to concentrate on the mammalian cytosolic enzymes for which there is a considerable body of biochemical data. However, over the last few years there has been increasing attention paid toward GSTs of other organisms. Structural studies of a Schistosoma GST are being pursued with the aim of defining the three-dimensional conformation of major epitopes, as a basis for improved vaccines against schistosomiasis [39]. This enzyme has been crystallized in the presence of reducing agent and S-hexylglutathione [39], conditions reminiscent of the bovine and human pi-class enzymes [30,32]. These conditions appear to be an absolute requirement for crystallization of all three proteins although the reasons for this are not clear. The inhibitor may be required to lock the enzymes into a particular conformational state or its detergent-like properties may be an important ingredient in the crystallization conditions. We have recently obtained crystals of a GST from Lucilia cuprina, the Australian sheep blowfly (unpublished data). It is thought that GSTs play an important role in the acquisition of resistance to insecticides [27,69,70] and thus insect GSTs represent a target for rational design of inhibitors to enhance the effectiveness of insecticides. Of particular interest is the finding that the amino-acid sequences of insect GSTs are related to theta-class GSTs (Table 1). Thus elucidation of the insect GST structure would establish the general fold of the theta-class enzymes.

7. Heterodimer formation To date there are no crystal structures available for a GST heterodimer. However, it is possible to speculate with some confidence on the likely structures of heterodimers given the high degree of sequence identity commonly found within each isoenzyme class. Jones and coworkers [40] have analysed the available alphaclass sequence data. They found that the domain and dimer interfaces tend to be strictly conserved as a general rule. Thus the structures of alpha-class homoand heterodimers are expected to be very similar. This proposal is supported by the observation that crystals of alpha-class homo- and heterodimers appear isomorphous with each other [31,33]. There was one exception to the rule found in an analysis of the four available rat sequences. In this case, one GST exhibited a lower sequence identity (60%) with the others and some of the changes mapped into the dimer interface area. Nevertheless, the changes were found to be conservative and at most, were expected to cause only small rigid body adjustments at the dimer interface.

11

A similar analysis of rat and mouse mu-class sequences shows that not all residues in the domain and dimer interfaces are strictly conserved but nevertheless the changes tend to be conservative (unpublished results).

8. I~cation of the non-substrate binding site The location of the non-substrate binding site has not yet been determined for any GST. Various biochemical and kinetic studies have been performed with the aim of identifying the site. Early studies suggested GSTs possessed a single binding site per dimer for heme, bilirubin and bile acid [72]. Studies on the binding of hemin to pi-class GST demonstrated that there was both a high and low affinity binding site [73]. It was shown that bilirubin could also bind to the high affinity site. Both sites appeared distinct from the GSH and substrate binding sites since hemin still bound to them in the presence of S-methylglutathione. This result was further supported by studies using bromosulfophthalein which bound equally well to mutants in which the C-terminal region of GST was truncated, whereas the sensitivity of the substrate binding site towards competitive inhibitors was dramatically reduced [74]. However, the non-substrate binding site may not be too far away from the GSH and substrate binding sites. GSTs are often inhibited upon binding of non-substrate ligands. Electron paramagnetic resonance measurements of a nitroxide-labelled human placental enzyme titrated with hemin suggests the binding site is about 10 ,~ away from Cys-47 [75]. Cys-47 is located 10.7 ,~ away from the glutathione sulfur atom. A number of residues have been implicated to be in the binding site by biochemical studies. The binding site for bromosulfophthalein may involve a tryptophan-containing peptide in the N-terminal region based on photoaffinity techniques [76]. The binding site for endogenous fatty acids has been traced to the peptide region 141 to 188 of a rat pi-class GST using fluorescence-labelling techniques [77]. The binding site accommodated fatty acids of varying chain lengths (12 to 18 carbons) and is probably also the bilirubin binding site [78]. It is tempting to speculate that large ligands may bind in the large V-shaped cleft formed at the subunit interface (Fig. 3A and B) although the interface appears too polar to bind very hydrophobic ligands.

9. Conformational changes Structural studies of enzymes have highlighted the importance of conformational flexibility and movement in the catalytic mechanism. In many cases the active

M.C.J. Wilce, M. IV.. Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

12 60

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Fig. 9. C o m p a r i s o n of the a v e r a g e d m a i n - c h a i n t e m p e r a t u r e factors for the G S T crystal structures. T h e r e is a s e p a r a t e trace for each m o n o m e r of the mu-class and pi-class enzymes.

to suggest it may be shielded in the absence of ligand. The pi-class GSTs can be reversibly inactivated by oxidizing agents with a concomitant formation of intraand inter-subunit disulfide bonds [30,84]. The inactivated enzyme loses its affinity for glutathione. A disulfide bridge between residues Cys-47 and Cys-101 has been identified in the oxidized form of the human placental pi-class enzyme [85]. These cysteines are 18.8 apart in the crystal structure of the reduced enzyme suggesting major conformational differences between the oxidized and reduced states. This possibility is supported by CD, UV and fluorometric analyses which demonstrate that some aromatic residues become more exposed in the oxidized state, although there is no change in secondary structure [85]. Preliminary model building and simulations suggest that helix a 2 folds into the substrate binding site in the oxidized state (unpublished results). This leads to the hypothesis that helix a 2 might normally sit in the substrate binding

A

site is quite hydrophobic in nature, and in the absence of ligand, tends to be shielded from solvent. Evidence has been obtained for a number of enzymes for the presence of a lid that covers the active site and opens or closes in response to ligand binding [79-82]. We have seen in the case of GSTs that the active site is, in part, composed of relatively flexible regions: notably the regions about helix a 2 which line part of the GSH binding site, the mu-loop and the C-terminal tail which impinges into the substrate binding site (see Fig. 9). Unfortunately, there are no crystal structures available for an unligated form of a GST, but there is some indirect evidence that the substrate binding site is at least partially buried in the absence of glutathione. Examination of the substrate binding site in the three different crystal structures suggests the Cterminus could play a potential role in shielding at least part of the substrate binding site from solvent. The 7 hydrophobic residues that line the substrate binding site of the pi-class GST create a patch of approx. 240 ,~2 of hydrophobic surface area. A lower estimate of the energy gained by burying this surface is approx. 25 cal.~ -2 mo1-1 (see [83]). In the alpha-class enzyme, the C-terminal helix buries a considerable area of the substrate binding site from solvent and in doing so, introduces new hydrophobic surface contributed from a face of the helix; which packs onto the substrate binding site to create a hydrophobic cavity (Fig. 10E,F). In the mu-class enzyme the C-terminus folds into a loop-like structure sitting over the substrate binding site to create a hydrophobic tunnel shielded from solvent (Fig. 10C,D). In the pi-class enzyme the substrate binding site appears totally exposed to solvent (Fig. 10B) but there is some evidence

B

Cysteinyl

D

I

Fig. 10. T o p o l o g i c a l r e p r e s e n t a t i o n s of the s u b s t r a t e b i n d i n g site. S u b s t r a t e b i n d i n g site a n d n e i g h b o u r i n g r e s i d u e s are r e p r e s e n t e d in C P K fashion. I n h i b i t o r s are shown as ball-and-stick. (A) g l u t a t h i o n e (B) S - h e x y l g l u t a t h i o n e b o u n d to h u m a n pi-class GST. (C) g l u t a t h i o n e b o u n d to rat mu-class GST. (D) as in (C) but C - t e r m i n a l r e s i d u e s r e m o v e d for clarity. (E) S - b e n z y l g l u t a t h i o n e b o u n d to h u m a n alphaclass GST. (F) as in (E) but C - t e r m i n a l r e s i d u e s r e m o v e d for clarity.

M.C.J. Wilce, M.W. Parker /Biochimica et Biophysica Acta 1205 (1994) 1-18

site in the unligated state because, unlike the other GST classes, the substrate binding site is more exposed and it would be energetically more favourable to bury its hydrophobic face from the aqueous environment. As an aside, these results suggest that the activity of the pi-class enzyme might be regulated by redox potential in vivo. Recent work suggests inactivation of GST through oxidation in vivo can be countered by the action of a thioltransferase [86]. Further indirect evidence of conformational changes comes from analysis of various kinetic data that is best explained in terms of such changes [22,87]. Direct evidence of any conformational changes may come from the structural studies of GST crystals grown in the absence of ligands [33]. An alternative approach of probing conformational changes through the use of monoclonal antibodies has being considered, A first step in this direction has been described by Fahl and coworkers who have generated a panel of antibodies directed towards the pi-class enzymes [88].

I0. Chemical modification studies

Prior to the publication of the crystal structures, chemical modification studies were a favoured way of pinpointing residues that might be involved in the catalytic mechanism of GSTs. In hindsight, it turns out that the results of most of these studies were misleading. It is worth briefly discussing some of these studies as the crystal structures suggest why misleading results were obtained in some cases. Cysteine residues have been implicated in the catalytic activity of pi-class GSTs by a number of groups based on the observation that thiol-reactive agents cause inhibition [30,89-92]. The pi-class structures show that there are no cysteines in either the GSH or substrate binding site [35,37]. Furthermore, site-directed mutagenesis of the cysteines showed they were not involved in catalysis [93-95]. Chemical modification of various cysteine mutants showed that modification of Cys-47 was responsible for the inhibition and the enzyme was only inhibited by large modifying reagents, such as N-ethylmaleimide [95]. These results can now be explained in light of the crystal structure. Cys-47 is located on the surface of domain 1 with its thiol group pointing into a small hydrophobic pocket (Fig. 11). Some of the main chain atoms of Lys-44 and Gin-51 and the side chain atoms of Trp-38 and Leu-52 form part of the inner wall of the pocket. The opposite face of this wall comprises part of the GSH binding site where all these residues are involved in contacts with bound glutathione (Fig. 4A). Chemical modification of Cys-47 with a bulky reagent would disturb the structure of the inner wall leading to the repositioning of key residues that recognize and bind glutathione.

13

G-site

H-site Fig. 11. View of the microenvironment around Cys-47 in the human placental pi-class GST crystal structure. The inhibitor, S-hexyl glutathione, is shown in ball-and-stick fashion with the glutathione moiety in the GSH binding site and the hexyl tail in the substrate binding site of the enzyme. Secondary structural elements are denoted by solid arrows for beta-strands and coiled ribbons for helices.

Similarly, cysteines have been implicated in the activity of the mu- and alpha-class GSTs. A direct role of cysteines in inhibition was subsequently eliminated by site-directed mutagenesis studies [96-99]. Chemical reaction with the glutathione analogue S-(4-bromo-2,3dioxobutyl)glutathione and analysis of subsequent proteolytic digests suggested Cys-ll2 in an alpha-class GST was in or close to the substrate binding site [100]. In the crystal structure, Cys-ll2 lies on the surface of domain 2, adjacent to the substrate binding site. However, the proceeding residue, Val-lll, does form part of the lining of the substrate binding site so the chemical modification studies have correctly identified Cys112 as being in the vicinity of the substrate binding site. There is an intriguing alternative interpretation of the results. The sidechain of Cys-ll2 points into the dimer interface in an area adjacent to the GSH binding site. The same area was conjectured to be a candidate substrate binding site in the original crystallographic studies of the pi-class GST from pig lung [35]. Perhaps GSTs have multiple substrate binding sites as a means of increasing their capacity to recognize structurally diverse substrates. Histidine residues have been implicated in catalysis on the basis of chemical modification studies [92,101]. There are no histidines in the active sites of any of the GST crystal structures and site-directed mutagenesis have largely confirmed they are not involved in catalysis [93,98, 102-104]. The most conserved histidine residue in the mu-class GSTs, His-15, has been subjected to site-directed mutagenesis [102]. The specific activity of five different mutants was considerably lowered but there was little alteration in the binding affinity for S-hexylglutathione. Although His-15 is lo-

14

M.C.J. l~lce, M.W. Parker /Biochimica et Biophysica Acta 1205 (1994) 1-18

cated approx. 15 A away from the GSH binding site, it appears to fulfil an important structural role as it is positioned in a hydrophobic pocket located at the domain interface. The human placental pi-class enzyme can be inactivated by the binding of pyridoxal 5'-phosphate at Lys127 suggesting this residue played a role in catalysis [105]. The closest distance between the lysine and bound glutathione is about 15 ,~ with the lysine being contributed from the twofold-related subunit. The lysine is positioned at the edge of the dimer interface, so it is feasible that pyridoxal binding causes local disruption of the interface with the effect being transmitted to the neighbouring GSH binding site resulting in inhibition. In most cases, site-directed mutagenesis studies have been successful in identifying those results from chemical modification studies that were misleading. However, even site-directed mutagenesis results have sometimes proved misleading. For example, two highly conserved arginines appeared to be located in the active site of an alpha-class GST based on the behaviour of single-site mutants [87]. The crystal structure showed that neither residue were located in the active site and the behaviour of the mutants was ascribed to secondary effects [40]. When Asp-98 of a pi-class GST was replaced by alanine, the K m was sixfold larger than wild-type [106], but barely different when replaced by asparagine [107]. The different results were attributed to the choice of amino-acid replacement [106].

11. Catalytic mechanism

The steady-state kinetic mechanisms of GSTs from the major classes have been studied and several mechanisms have been proposed including ping-pong, sequential and random mechanisms (for example, see Refs. 108-110). The majority of investigations suggest the order of glutathione and substrate addition is random. Under physiological conditions, the reaction would be ordered with glutathione adding first, given that the concentration of glutathione in normal cells (1-10 mM) is about three orders of magnitude higher than the dissociation constant between glutathione and enzyme. The specificity of the GSH binding site for glutathione is high. Early studies demonstrated the importance of interactions with the y-glutamic moiety of bound glutathione through a series of binding studies with synthetic glutathione analogues [111,112]. The multiple interactions between the y-glutamyl group and the protein seen in the crystal structures explain these observations. It was also found that the orientation of the glutathione thiol group was critical. The glycyl group was much less restrictive and thus less

important for substrate recognition [118]. Surprisingly, not all glutathione-contacting residues are conserved between the GST classes, although substitutions have tended to be conservative (Fig. 4A-C). The results of site-directed mutagenesis have largely supported the crystallographic results and confirmed the importance of residues that have been identified in the GSH binding site [93,113-117]. A central aspect of the catalytic mechanism is the lowering of the pK a of the glutathione thiol group from 9 in aqueous solution to between 6 and 7 when bound to the protein [67,112,119]. The crystal structures suggest a number of ways the thiolate anion might be stabilized. The helix dipole of a l is positioned so that it may contribute to the stability of the anion. In all crystal structures examined so far, there is the same conserved tyrosine within hydrogen-bonding distance of the thiol sulfur. Mutagenesis studies have shown that the conserved tyrosine is responsible for activation of glutathione by acting as a hydrogen bond donor and thereby promoting thiolate formation [116,120-123]. Armstrong and coworkers have suggested that this tyrosine may play additional roles such as correctly orientating the glutathione in the active site and facilitating the passage of the thiol proton out of the active site [123]. In the alpha-class enzyme the thiolate anion appears to be further stabilized by Arg-15 which is within hydrogen-bonding distance of the sulfur atom. The mutation of Asp-98 to alanine increases the pK a of the thiol group by about 0.8 units which supports an additional role for Asp-98 in facilitating the formation of the thiolate anion [106,122]. Direct evidence has been obtained for the rate-limiting formation of a Meisenheimer complex in GSTcatalysed nucleophilic aromatic substitution reactions [67]. It is not obvious how the intermediate is stabilized, although it has been speculated that the helix dipole of a l is one possible source of stabilization [54].

12. Comparison to other glutathione-binding enzymes

Mannervik had speculated in 1985 that GSTs may share an ancestral gene that coded for a glutathione-binding protein [9]. It was perhaps then not surprising to find the topology of domain 1 of GSTs resembled the structures of a number of glutathione-binding proteins; namely E. coli thioredoxin [124], T4 glutaredoxin [125] and glutathione peroxidase [126], despite the lack of any detectable sequence homology (Fig. 12). Sinning et al., [40] have also identified a similarity with glutathione reductase [127] although there is one change in the topology with the equivalent of helix a3 preceding the first strand rather than the fourth strand. The matches are so striking that it has prompted Sinning et al., [40] to describe the N-terminal domain as a canoni-

M.C.J. Wilce, M. 14I..Parker/Biochimica et Biophysica Acta 1205 (1994) 1-18

A

15

cal glutathione-binding domain. These analyses are supported by the finding that a peptide fragment consisting only of the N-terminal domain retains the capacity to bind glutathione [88]. However, the significance of these matches should be considered in the light of the fact that there are no residues conserved in all the structures. This might be explained on the basis that the sequences of the glutathione-binding domains have diverged so far from the ancestral gene that encoded the domain, that any sequence similarity has been lost. However, it seems unlikely that all the key residues participating in glutathione-binding would be substituted for in the course of evolution. The alternative hypothesis of convergent evolution should be considered in which the domain topology represents a stable fold that has been adapted by a number of proteins for the binding and utilization of a prevalent ligand.

B 13. Basis for substrate selectivity--the substrate binding site The remarkable tolerance of GSTs for a diverse range of substrates can be attributed in part to the large number of different isoenzymes from within and between classes. Even so, individual members still show a large degree of tolerance. The overall size and shape of the substrate binding site varies considerably between GSTs as shown in Fig. 10 and are likely to be important determinants of specificity. The extent of inhibition has been shown to correlate with the length of the alkyl side chain of S-substituted glutathione inhibitors [68,128,129]. The substrate specificities of fifteen GSTs for 4-hydroxyalkenals showed no correlation with structural class but highlighted the importance of the degree of hydrophobicity and steric limitations of the substrate binding site [129]. Some of the inter-class differences can readily be ascribed to the structural variations of the C-terminal residues observed in the crystal structures of the different GSTs. Photoaffinity labelling studies [130] and deletion mutants [74] have implicated the C-terminal region of GSTs in substrate selectivity. The C-terminal tail which forms part of the substrate binding site in all three GST crystal structures, is longer in the alpha- and mu-class enzymes. The extra extension in the alpha-

Fig. 12. Ribbon representations of protein domains bearing some topological resemblance to domain 1 of GSTs. Residues were assigned to secondary structural elements according to criteria defined by Kabsch and Sander [133]. The figures were produced by the computer program MOLSCRIPT [134]. (A) Domain 1 of pi-class GST. (B) Thioredoxin from E. coli [124]. (C) glutathione peroxidase [125].

16

M. C.J. Wilce, M. W. Parker / Biochimica et Biophysica Acta 1205 (1994) 1-18

class enzyme adopts a helical fold with the hydrophobic face of the helix forming part of the substrate binding site wall. As a consequence, the substrate binding site becomes enclosed by the wall and is smaller and more hydrophobic than the other two classes (Fig. 10E,F) [40]. The origin of the distinctive intra-class substrate specificities observed in GSTs is, at least in part, due to often subtle changes in the substrate binding site. The residue differences between alpha-class sequences have been m a p p e d onto the h G S T A I - 1 crystal structure [40]. For the most homologous sequences, about one third of the changes were localized to the substrate binding site. The substitutions either changed the size and shape of existing residues (e.g., Met to Leu or Ala to Val) or introduced some hydrogen-bonding functionality (e.g., Phe to Ser or Ala to Ser). In one extreme case, a hydrophobic residue was substituted for a charged residue although the residue length was approximately maintained (Met to Glu). Another source of substrate specificity may lie in the degree of flexibility of the C-terminal tail which projects into the substrate binding site. Construction of chimaeric enzymes appears to be a promising approach for the rapid identification of key catalytic residues in the active sites of distantly related enzymes. In this method, whole regions of low sequence homology can be exchanged by modular mutagenesis and the behaviour of the chimaeric protein evaluated. This approach could be particularly useful in the case of GSTs where there is such a high degree of sequence diversity in the substrate binding site making it difficult to pinpoint which residues are important for substrate specificity on the basis of sequence alignments alone. Armstrong and coworkers [54] have used the method to probe the molecular basis for substrate preferences in the rat mu-class isoenzymes 3 - 3 and 4-4. The 3 - 3 isoenzyme is better at catalysing nucleophilic aromatic substitution reactions whereas the 4 - 4 isoenzyme is better at catalysing the conjugation of glutathione to oxiranes and a,/3-unsaturated ketones. Regions of variable sequence in the 3 - 3 enzyme were replaced by the equivalent regions in the 4 - 4 enzyme. The behaviour of the resulting constructs suggested that a point mutation at Val-9 was a critical residue for determining the stereoselectivity of the two isoenzymes.

14. Future directions X-ray crystallographic investigations over the last few years have revealed a common protein fold for cytosolic GSTs and lead to detailed descriptions of the active sites. The role of the conserved tyrosine has been highlighted and the direct participation of histidine and cysteine residues in the catalytic mechanism

eliminated. In spite of the wealth of knowledge generated by the crystal structures, many questions remain unanswered. In particular, many of the details concerning the catalytic mechanism are yet to be elucidated. Further progress can be expected in the near future as it is now possible to rationally design mutants and inhibitors using the crystal structures as a guide. Studies of the evolutionary relationships between GSTs would be greatly aided by the determination of nonmammalian G S T structures. A detailed understanding of G S T function at the molecular level will lay the basis for the rational design of therapeutic agents in the future.

15. Acknowledgements We thank Dr. Alwyn Jones, Prof. Bengt Mannervik and coworkers for providing us with the alpha-class G S T coordinates and a preprint describing their latest results. We thank Dr. Gary Gilliland for making the mu-class coordinates available to us through the Protein Data Bank. We would like to acknowledge the valuable contributions of Drs. Mario Lo Bello, Peter Reinemer, Rudolf Ladenstein and Profs. Giorgio Federici, Robert H u b e r towards the structural studies of human placental pi-class GST. M.W.P. is a Wellcome Australian Senior Research Fellow. This work was supported by grants from the Australian Research Council and the National Health and Medical Research Council.

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