2
Chem. Res. Toxicol. 1997, 10, 2-18
Invited Review Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases Richard N. Armstrong* Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received April 25, 1996
Introduction The discovery in 1961 of a catalytic activity for the addition of glutathione (GSH) to 1,2-dichloro-4-nitrobenzene in cytosolic extracts of liver (1, 2) initiated three decades of increasing interest in the genetics and enzymology of the glutathione transferases (EC 2.5.1.18), culminating in the elucidation of the three-dimensional structures of several cytosolic isoenzymes in the first half of this decade. The structural information has had a profound impact on our understanding of the catalytic mechanisms of the glutathione transferases, the evolution of the protein fold, and the molecular basis for their participation in the detoxification of endogenous and xenobiotic electrophiles. The purpose of this review is to illustrate how the knowledge of the three-dimensional structures has influenced current ideas on the evolution and function of these enzymes in biological systems. A number of review articles covering various aspects of the biochemistry, structure, and gene regulation of the GSH transferases (3-12) are available. A satisfactory understanding of the biological function and significance of the GSH transferases requires a knowledge of where the enzymes came from, how they work, and how their expression is regulated. The glutathione transferases catalyze the general reaction shown:
GSH + R-X f GSR + HX
(1)
The issues with respect to how these enzymes function can be framed by two general categories of questions addressing, on the one hand, how the enzyme recognizes and activates glutathione for nucleophilic attack and, on the other, how, or even if, the enzyme specifically recognizes electrophilic substrates (R-X). This division is not entirely arbitrary, as will be seen from a description of the structures below. There are both cytosolic and microsomal GSH transferases that are involved in the metabolism of xenobiotics. The cytosolic or soluble enzymes derive from a superfamily of genes that encode at least six individual classes of enzymes, five of which are known to be represented in vertebrates, as illustrated in Table 1. The soluble enzymes defined to date are invariably either homodimeric or heterodimeric species. However, heterodimers form only between subunits of the same enzyme class. Threedimensional structures of at least one representative of five of the enzyme classes are known. The structures have revealed basic features of each enzyme class which * Phone: (615) 343-2920; FAX: (615) 343-2921; email: armstrong@ toxicology.mc.vanderbilt.edu.
S0893-228x(96)00072-0 CCC: $14.00
are invaluable in tracking the evolution of the structural and catalytic features of the enzymes. For example, both the subunit interface and the active site residues involved in the ionization of GSH have evolved considerably though not in tandem. Unfortunately, the nomenclature for the various enzyme classes from species other than humans remains unsettled. A nomenclature has been agreed upon for the human cytosolic enzymes which is, in principle, applicable to all vertebrate glutathione transferases and extendible to prokaryotes and other organisms (13). The human enzymes are named with respect to the class in which they fall (A, M, P, K, or T for alpha, mu, pi, kappa, and theta) with their subunit composition or isoenzyme type designated by Arabic numerals. For example, a homodimer of type 1 mu subunits is M1-1 and the heterodimer of type 1 and 2 alpha subunits, A1-2. Allelic variants of isoenzymes are designated by lower case letters following the subunit type (e.g., M1a-1b for the class mu hybrid of the 1a and 1b alleles). The nomenclature used in this review follows the system agreed upon for the human enzymes. Three membrane-bound glutathione transferases are known, one of which appears to be involved in xenobiotic metabolism. Microsomal GSH transferase I is an integral membrane protein that has been characterized from both rats and humans where it is found in large amounts in liver and is distributed in both the microsomal and outer mitochondrial membrane (14). The protein bears no discernible relationship to any of the known cytosolic enzymes with respect to sequence. However, it does appear to be vaguely similar to two other membranebound GSH transferases, leukotriene C4 (LTC4) synthase and microsomal GSH transferase II, in that the three proteins are about the same size, share a small amount of sequence identity, and are all membrane-bound (15, 16). Leukotriene C4 synthase and microsomal GSH transferase II are the only mammalian glutathione transferases which have a clearly defined physiologic substrate, leukotriene A4. There is no apparent relationship between the LTC4 synthases and the cytosolic enzymes (16-18). However, both LTC4 synthase and microsomal GSH transferase II show a remarkable degree of sequence identity to the 5-lipoxygenase activating protein as well as the small degree of sequence similarity with the microsomal GSH transferase I noted above. Considerably less is known about structure and catalytic mechanisms of the membrane-bound glutathione transferases. Finally, there is one known plasmid-encoded bacterial glutathione transferase that is associated with bacterial © 1997 American Chemical Society
Invited Review
Chem. Res. Toxicol., Vol. 10, No. 1, 1997 3
Table 1. General Features of Glutathione Transferases Inferred from Sequences, Physical and Catalytic Properties, and Crystal Structures molecular mass, monomer (kDa)
quaternary structure
alpha pi mu sigmab theta kappa
26 23 26 23 27 25
dimer dimer dimer dimer dimer dimer
frpd
16
dimer
microsomal I microsomal II LTC4 synthase
17 17 17
trimer ? ?
enzyme class
subunit interface
Cytosolic/Soluble ball/socket ball/socket ball/socket hydrophilic hydrophilic ?
catalytic type
three-dimensional structures known
Tyr/Arg Tyr Tyr Tyr Ser ?
human/rat human/mouse/pig human/rat/S.j.a squid blowfly/A.t..c none
Metal-Dependent none Membrane rat, 2D projection none none
a Schistosoma japonicum. b A class sigma enzyme has not yet been identified in vertebrates. c Arabidopsis thaliana. resistance protein.
resistance to the antibiotic fosfomycin, (1R,2S)-1,2-epoxypropylphosphonic acid (19, 20). This enzyme is quite an unusual GSH transferase in that it is highly specific for the addition of GSH to the oxirane ring of fosfomycin and appears to be a metalloenzyme (20).1 Its primary structure is not related to any of the soluble or microsomal GSH transferases, nor does it catalyze the addition of GSH to any of the usual electrophilic substrates (e.g., CDNB) used to assay the soluble enzymes (20).
Three-Dimensional Structures of the Cytosolic GSH Transferases Subunit Structures. The three-dimensional structures of at least one member of five of the six principal enzyme families (alpha, mu, pi, sigma, and theta) have been determined (21-34). Enzymes in complex with GSH, GSH analogues, products, and transition state analogues as well as the unliganded enzyme are represented. All of the structures have the same basic protein fold which consists of two domains, as illustrated in Figure 1. The overall fold of the N-terminal domain is classified as part of the thioredoxin superfamily fold, which also includes glutaredoxin, disulfide-bond formation facilitator, and glutathione peroxidase (35). This domain constitutes roughly one-third of the protein and consists of a β-R-β-R-β-β-R structural motif that forms a mixed four-strand β-sheet in the order of 4312 with strand 3 antiparallel to the others. The core of the domain is composed of three layers with the β-sheet sandwiched between R-helices (R/β/R). The C-terminal two-thirds of the protein is an all-R-helical domain with a unique protein fold, the core of which consists of a bundle of four helices. The primary function of the N-terminal domain is to provide the binding site for GSH, as illustrated in the representation of the product complex in Figure 2. The peptidyl portion of the product lies at the end of the β-sheet and interacts with the protein via a number of electrostatic and hydrogen-bonding interactions, as discussed in more detail below. The xenobiotic moiety of the product is located in the crevice between the two domains and makes a number of contacts with residues in domain II, particularly along the face of the R4-helix and the C-terminal tail. Thus, domain II appears to provide structural elements for the recognition of xeno1B.
Bernat and R. N. Armstrong, unpublished results.
d
Fosfomycin
biotic substrates and helps to define the substrate selectivities of the various isoenzymes. The specific roles for particular residues in substrate recognition and catalysis are discussed in more detail in the following sections. Domains I and II are often loosely referred to as the GSH and xenobiotic substrate binding domains, respectively. In spite of similar overall topologies, the structures differ considerably with respect to a number of details. The most notable topological differences include the muloop and the R9-helix of the class mu and alpha enzymes, respectively. Both of these structural elements are located adjacent to the substrate binding sites and contribute to a more constricted active site of these two enzyme types when compared to examples of pi, sigma, and theta classes. The features of subunit-subunit recognition also differ between enzyme families. In addition, many of the groups involved in the binding of GSH have been altered through evolution. Quaternary Structure. The subunits in the dimeric enzyme are related by a twofold axis (C2 symmetry), as shown Figure 3. The principal intersubunit interactions occur between domain I of one subunit and domain II of its partner. The subunit interface of the class alpha, mu, and pi enzymes is characterized by a ball-and-socket hydrophobic interaction that is established by wedging the side chain of a phenylalanine residue (the ball) (F52, alpha; F56, mu; F47, pi), which protrudes from a loop between the R2-helix and strand β3 of domain I of one monomer, into a hydrophobic socket located between the R4- and R5-helices of domain II of the other monomer, as illustrated in Figure 3. This particular interaction is not observed in the class sigma and theta enzymes. The dimer interface for the class theta and sigma enzymes differs in that the phenylalanyl residue and the loop on which it resides are absent and that the hydrophobic socket between helices 4 and 5 is not present (Figure 3). Heterodimers derived from subunit types from different enzyme classes are not observed due incompatible or suboptimal protein surfaces. This is particularly true for subunits representing the two basic types of subunit interfaces noted above. The structural basis for the familial recognition in the more closely related alpha, mu, and pi class enzymes appears to be related to differences in interdomain rotations in the subunits. Domains I and II have slightly different orientations with respect to one another in these three enzyme classes such that the surface of one enzyme class is not completely compatible
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Chem. Res. Toxicol., Vol. 10, No. 1, 1997
Armstrong
Figure 1. Ribbon diagrams of the three-dimensional structures of the class alpha, mu, theta, pi, and sigma GSH transferases in complex with S-benzylglutathione, (9S,10S)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene, S-hexylglutathione, glutathione sulfonate, and 1-(S-glutathionyl-2,4-dinitrobenzene, respectively. The views are of a single subunit perpendicular to the twofold axis of each dimer. The unique structural elements, the R9-helix in the class alpha and the “mu loop” in the class mu enzymes can be seen in the middle and right side of the respective structures. The diagrams were made with the program MOLSCRIPT (36) using the PDB files 1GUH (alpha), 2GST (mu), 1GSR (pi), and 1GSQ (sigma). The coordinates for the class theta enzyme were provided by Dr. Michael Parker and colleagues. The hexyl portion of S-hexylglutathione and residues 202-208 are not visible in the electron density map of the class theta structure.
with another (24). Folding, Subunit Assembly, and Cooperativity of the Dimer. Two questions that constantly come up are: Why are the GSH transferases dimers? and: Is there any evidence for a catalytically active monomer? One reason these questions are so often asked is that there are, as yet, no conclusive answers. Catalytically active monomers have not been observed, but perhaps the appropriate conditions have not been found. The few denaturation studies that have been performed on the cytosolic enzymes tend to suggest that unfolding, as monitored by both structural and functional probes, is best described as a two-step process (38). Results with the class pi isoenzyme suggest that the dimeric structure stabilizes the tertiary structure of the individual subunits and presumably each of the two domains of the subunit (39). Whether this is a general property of all soluble GSH transferases remains to be determined. There is no evidence for either positive or negative cooperativity in native dimeric GSH transferases. However, mutants of the class pi enzyme involving disruption of an ion pair between Cys47 and Lys54 exhibit positive cooperativity with respect to CDNB activity and GSH binding (40, 41).
Binding and Activation of GSH The GSH Binding Site. Perhaps the most fundamental aspect of the catalytic mechanism is how the
enzyme uses the binding interactions with GSH to activate the sulfur for nucleophilic attack. The peptide is bound in an extended conformation, with the γ-glutamyl residue pointing down toward the dimer interface, the cysteinyl sulfur pointing to the subunit to which it is bound, and the glycyl residue residing near the surface of the protein. The molecule is anchored by over a dozen electrostatic interactions utilizing virtually all of the hydrogen bond donor and acceptor sites on the peptide. Even though the orientation of the peptide in the active site is approximately the same for all of the isoenzymes, there are substantial differences in the details of the hydrogen-bonding interactions. The differences in the hydrogen-bonding patterns for GSH binding to the various isoenzymes have been discussed elsewhere (9-12) and will not be reiterated here except where they are directly relevant to catalysis. The most conserved region of structure in all of the cytosolic enzymes is a core ββR motif that is responsible for recognition of the γ-glutamyl portion of the peptide. This structural element, illustrated in Figure 4, commences with a cis-proline residue just prior to β3 and continues through the R3-helix and supplies hydrogen bonding partners that recognize the amino and carboxylate groups at the chiral R-carbon of the γ-glutamyl residue. These interactions involve two residues located in the turn between strand β4 and the R3-helix, a
Invited Review
Chem. Res. Toxicol., Vol. 10, No. 1, 1997 5
Figure 2. RASTER3D (37) representation of one subunit of a class mu isoenzyme (M1-1) from rat. The R-helices and β-strands are illustrated as cylinders and arrows, respectively. Domains I and II and the GSH conjugate, (9S,10S)-9-(S-glutathionyl)-10-hydroxy9,10-dihydrophenanthrene, are shown in yellow, blue, and red, respectively. The dotted lines represent hydrogen-bonding interactions between the hydroxyl group of Y6, located in domain I, and the sulfur of the conjugate and between the hydroxyl group of Y115, located at the end of the R4-helix of domain II, and the 10-hydroxyl group of the product.
glutamine or glutamate residue followed by a serine or threonine. The cis-prolyl residue that precedes this region helps to conserve the overall fold of the domain. The only other highly conserved interaction is between the carbonyl and NH groups of the cysteinyl residue and the main chain of the protein just preceding the conserved cis-prolyl residue. The similarity in this region of structure for the two most divergent classes of cytosolic enzyme, theta and alpha, is obvious and much more highly conserved (Figure 4) than are the interactions between the proteins and the sulfur of GSH (Figure 5). The most fundamental difference among the GSH binding sites of the various enzyme classes involves the interaction of the protein with the sulfur of the peptide, as illustrated in Figure 5. The theta class enzymes, thought to be the evolutionary precursor of the alpha, mu, pi, and sigma class proteins, appear to utilize the hydroxyl group of a serine residue located near the N-terminus of the polypeptide to activate the sulfhydryl group of bound GSH (32, 42). In contrast, the class alpha, mu, pi, and sigma enzymes have recruited the hydroxyl group of a tyrosyl residue, located in a slightly different position, to act as a hydrogen bond donor to the sulfur which lowers the pKa of the thiol in the E‚GSH complex so that it is predominantly ionized at physiological pH (31, 43-45). Finally, the class alpha enzyme has recruited yet another residue into the inner coordination sphere of the sulfur, the side chain of Arg15, as illustrated in Figure 5 (46). The reactive species in the binary complexes in all cases is most probably the thiolate anion (E-O-H----SG), which accepts a hydrogen
bond from the seryl or tyrosyl hydroxyl group and gathers additional stabilization from positive charge of Arg15 in the instance of the class alpha enzymes. Activation of the Nucleophile: Where Are the Protons? Irrespective of the general agreement concerning what residues are directly involved in the activation of GSH, there remains some dispute as to the actual role of the active site hydroxyl group in catalysis. The difference of opinion is over the position of protons in the active site. That is, does the hydroxyl group act as an electrophilic participant (hydrogen bond donor) or as a general base (hydrogen bond acceptor) either in the binding of glutathione or in the reactant state binary complex? With respect to the binding of glutathione, the situation is illustrated in the following equations:
E-O-H + GS-H h E-O-H----SG + H+
(2)
E-O- + GS-H h E-O-H----SG
(3)
The arguments (47-50) that the active site tyrosyl residue may act as a general base for the removal of the proton as in eq 3 are based on the observation that the hydroxyl group in the class alpha enzyme has an abnormally low pKa (ca. 8.5) (46-48) and that the analogous group in the class pi enzyme may have a low pKa (49, 50). In contrast, the pKa of the hydroxyl group in the M1-1 enzyme is about 10 (51). Even in the case of the class alpha enzymes, far less than 50% of the tyrosyl side chain is ionized at neutral pH, suggesting that the situation in eq 3 represents, at best, a minor pathway. A
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Chem. Res. Toxicol., Vol. 10, No. 1, 1997
Armstrong
Figure 4. MOLSCRIPT (36) representation of the highly conserved core ββR motif which is responsible for the recognition of the γ-glutamyl residue of GSH. Glutathione and the side chains located at the turn between the β4-strand and R3-helix involved in hydrogen-bonding interactions with the R-amino and R-carboxyl moieties of the γ-glutamyl residue are illustrated in ball-and-stick. The two motifs represented are from what are considered to be the most divergent classes of cytosolic GSH transferases, the theta and alpha classes.
has a pKa of about 6.7 and that the thiolate anion is the predominant species in the active site at neutral pH, as indicated by the following equation (43, 52): Figure 3. View of the class mu and sigma dimers down the twofold axis relating the two subunits. The class mu dimer (top) is representative of the alpha/mu/pi type interface with the balland-socket interaction resulting from the intercalation of a phenylalanine residue (Phe56) from domain I between the R4and R5-helices of domain II from the opposite subunit. The sigma/theta type interface lacks this interaction and is represented by the class sigma dimer (bottom).
much stronger case can be made for the hydroxyl group acting as a hydrogen bond donor as in eq 2. This is clearly the situation with the M1-1 enzyme from rat and is probably true as well for the class theta enzymes which utilize an alkyl- rather than an aryl-hydroxyl group. Moreover, the fact that anionic analogues of glutathione such as γ-L-Glu-L-AspGly and glutathione sulfonate bind to the enzyme more avidly than does GSH argues against a significant ionization of the active site hydroxyl in unliganded enzymes (52). Where is the proton in the monoprotonated binary complex that represents the ground state for reaction with an electrophilic substrate? The pH dependence of kcat/KmCDNB for the reaction with 1-chloro-2,4-dinitrobenzene at saturating concentrations of GSH suggests that the reaction catalyzed by class mu enzymes depends on an ionization with an apparent pKa of about 6.3, a value considerably less than the pKa of 9.0 for GSH in aqueous solution (43, 53). Similar results have been reported with the class alpha, pi, and sigma enzymes (31, 44, 45, 54). Spectroscopic observation of the thiolate anion at 240 nm in the active sites of binary complexes of rat isoenzymes M1-1 and M2-2 provides direct evidence that the thiol
E-O-H‚GSH h E-O-H----SG + H+ pKa ≈ 6.0-7.5 (4) E-O-H‚GSH h E-O----H-SG + H+
(5)
A pH-dependent spectral signal for the enzyme-bound thiolate is also observed with the class theta enzyme from Escherichia coli albeit with a higher pKa ≈ 7.5.2 The alternative view, that the proton is located on the sulfur of GSH as illustrated in eq 5, has been proposed with regard to the class pi enzyme (49, 50) but has little experimental support. The difference in the scenarios described by eqs 4 and 5 is not trivial, since the monoprotonated species on the right represents the ground state for reaction of the binary complex with the electrophilic substrate. In the case of eq 5 (general-base catalysis), a proton transfer from sulfur to oxygen must occur between the ground state and transition state for reaction. Solvent deuterium isotope effects on kcat/KmCDNB for the addition of GSH to CDNB catalyzed by class alpha (55) and mu (56) enzymes are small and inverse (kH/kD ) 0.8-0.9) and similar to that seen for the chemical reaction of GS-(aq) with the same electrophile (55). This magnitude of solvent deuterium isotope effect is consistent with desolvation of a thiolate anion between the ground and transition state and not with a proton transfer between sulfur and oxygen. 2J. F. Parsons, L. T. Laughlin, and R. N. Armstrong, unpublished results.
Invited Review
Figure 5. Evolution of first-sphere interactions between the active sites of class theta, mu, and alpha enzymes and the sulfur of GSH. It is possible that the tyrosine residue often, but not always, found near the N-terminus of the class theta enzymes was recruited in the evolution of the protein to the other classes. The most recently evolved enzyme, class alpha, has an additional residue (Arg15) conscripted into the first sphere of the sulfur of GSH. The structures are MOLSCRIPT (36) representations of coordinates from PDB files, 1GST and 1GUH. The coordinates for the class theta enzyme were courtesy of Dr. Michael Parker.
An instructive model for general-base catalysis has been created by substitution of the unnatural amino acid 3-fluorotyrosine for all fourteen tyrosine residues in the M1-1 isoenzyme (56). The hydroxyl group of the 3-fluorotyrosyl residue at position 6 has a pKa ) 7.5, which is 2.5 log units lower than the native enzyme. The spectral properties and pH dependence of kcat/KmCDNB suggest that the tetradeca(3-fluorotyrosyl)-enzyme exhibits ionization behavior consistent with that described in eq 5 with an apparent pKa ) 6.0. In contrast to the native enzyme, the fluorotyrosyl mutant exhibits a large inverse solvent deuterium isotope effect of 0.5. Moreover, a proton inventory of the reaction shows that this isotope effect arises from a single reactant state proton with a fractionation factor of 0.55. Thus, the 3-fluorotyrosinate anion appears to act as a general base to abstract the proton from sulfur between the ground state and transition state, with the proton being essentially fully transferred in the transition state. That the native enzyme
Chem. Res. Toxicol., Vol. 10, No. 1, 1997 7
behaves in a quite different manner is strong evidence that general-base catalysis is not an apt description of the role of the tyrosine in the chemistry of the native enzyme (56). Dissection of Active-Site Electrostatics by Mutagenesis. Site-specific mutagenesis has been a valuable tool for assessing electrostatic contributions of various residues in the vicinity of the sulfur to catalysis. For the purposes of discussion, these contributions are classified as first-sphere and second-sphere effects for ligands that are directly coordinated to the sulfur and those that are distal to the sulfur, respectively (Figure 6). The most prominent first-sphere interaction is the hydrogen bond from the hydroxyl group of the active site tyrosine or serine residue mentioned above. Removal of the hydroxyl group as in the Y6F mutant of the M1-1 isoenzyme from rat causes a loss of the difference absorption band at 240 nm associated with the thiolate anion of E‚GS- and results in a shift of the apparent pKa of bound GSH from 6.2 in the native E‚GSH complex to about 7.8 in the mutant. This shift in pKa corresponds to a -2.2 kcal/mol stabilization energy provided to the thiolate by the hydroxyl group of Tyr6. The contribution of the hydroxyl group of the conserved tyrosine residue of alpha, pi, and sigma class isoenzymes has also been investigated with similar results (31, 44, 45, 54). The structural evidence strongly suggests that the function of the tyrosyl residue is replaced by serine in the class theta enzymes (32, 42). Although the exact mechanistic function of the serine hydroxyl group has not been fully evaluated, it appears that nature has evolved at least two different first-sphere interactions to stabilize the thiolate anion. Mutation of the tyrosine residue near the N-terminus of the presumed theta class enzyme from E. coli has little or no effect on catalysis (57). Interestingly, mutation of all of the other proton donors expected to be near the active site, including Cys10, Ser11, and Ser14, results in only small (
