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THE ENZYMES OF GLUTATHIONE SYNTHESIS: y-GLUTAMYLCYSTEINE SYNTHETASE

OWEN W. GRIFFITH, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226 and R. TIMOTHY MULCAHY, Department of Human Oncology, University of Wisconsin Medical School, Madison, WI 53792

C O N T E N T S

I. Introduction 11. Overview of GSH Biosynthesis

111. y-Glutamylcysteine Synthetase A. PURIFICATION AND PROTEIN CHEMISTRY B. CLONING, SEQUENCING AND EXPRESSION OF 7-GCS SUBUNITS

Eukaryotic y-GCS Catalytic Subunit (y-GCSh) y-GCS Regulatory Subunit (y-GCSI) y-GCSh and y-GCS, mRNA Expression Expression of Recombinant y-GCSh and y-GCSI in Mammalian Cells Expression of Recombinant y-GCSh and y-GCSl in E. coli

GSHl and Gcsl: The Yeast yGCS Genes The Human Catalytic Subunit (y-GCSh) Gene The Human Regulatory Subunit (y-GCS,) Gene

CHEMOTHERAPY

C. CLONING OF y-GCS GENES AND REGULATION OF EXPRESSION

D. ROLE OF y-GCS OVEREXPRESSION IN RESISTANCE TO CANCER

E. POSTTRANSLATIONAL REGULATION F. CATALYTIC MECHANISM

Substrate Specificity Reactions Catalyzed Steady State Kinetics Chemical Mechanism

Feedback Inhibition by GSH S-Alkyl Homocysteine Sulfoximine Inhibitors Clinical Use of BSO to Modulate GSH Levels in Cancer Patients Inactivation by Cystamine and Related Compounds Inactivation by Chloroketones Other Glutamate Antagonists Inhibition by Nitric Oxide Donors

G. INHIBITION

Advances in Enzymology and Related Areas of Molecular Biology, Volume 73: Mechanism of Enzyme Action, Part A, Edited by Daniel L. Punch ISBN 0-471-24644-1 01999 John Wiley &Sons, Inc.

209

Advances in Enzymology and Related Areas of Molecular Biology, Volume 73Edited by Daniel L. Punch

Copyright © 1999 by John Wiley & Sons, Inc.

210 OWEN W. GRIFFITH AND R. TIMOTHY MULCAHY

IV. Conclusions and Perspective V. Acknowledgments

References

I. Introduction

Glutathione (L-y-glutamyl-L-cysteinylglycine, GSH') is synthesized from its constituent amino acids by many prokaryotes of the eubacteria sub- kingdom and by virtually all eukaryotic cells (Entamoeba histolytica and Giardia are the main exceptions identified to date (Fahey et al., 1984; Mehlotra, 1996; Newton and Fahey, 1990; Fahey and Sundquists, 1990). Fahey and coworkers have argued that the ability to synthesize GSH probably originated in an ancestor common to the cyanobacteria and the photo- synthetic purple bacteria and then migrated to eukaryotes during the en- dosymbiotic process that resulted in chloroplasts and mitochondria (Fahey et al., 1984; Mehlotra, 1996; Newton and Fahey, 1990; Fahey and Sundquist, 1990). Consistent with this view, the enzymes of GSH synthesis are found mainly in plant chloroplasts (Hell and Bergmann, 1988, 1990; Lancaster et al., 1989; Klapheck et al., 1987) organelles that apparently originated from cyanobacteria (Gray, 1988). In animals, the ancestry of GSH synthesis is less evident. Analysis of ribosomal RNA (rRNA) sequences shows homologies between group IIa purple bacteria and mitochondria (Gray, 1988), but mitochondria in modem animals lack the enzymes of GSH synthesis (Griffith and Meister, 1985). Nevertheless, the prokaryotic and mammalian enzymes of GSH synthesis share several limited regions of amino acid sequence homology that support the Fahey hypothesis. Furthermore, significant similarities in catalytic mechanism and in response to inhibitors suggest an evolutionary link between the prokaryotic enzymes of GSH synthesis and those found in protozoa, plants, and animals.

' Abbreviations used: GSH, glutathione; GSSG, glutathione disulfide; y-GCS, y-glutamylcysteine synthetase; y-GCSh, y-GCS catalytic (heavy) subunit; y-GCS,, y G C S regulatory (light) subunit; L-PAM, melphalan (L-phenylalanine mustard); MSO, methionine sulfoximine; a-et-MSO, a-ethyl-methionine sulfoximine; PSO, prothionine sulfoximine; BSO, buthionine sulfoximine; P-NF, P-naphthoflavone; ARE, antioxidant response element; MRP, rnultidrug resistance protein; PKA, protein kinase A; PKC, protein kinase C; PBL, peripheral blood leukocyte; MDP, N-2-mercaptoethyl-l,3-diaminopropane; RSNO, S-nitrosothiol; HPLC, high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

THE ENZYMES OF GLUTATHIONE SYNTHESIS 21 1

In the animal kingdom, the ability to synthesize GSH is apparently uni- versal, and GSH is the predominant low-molecular-weight thiol in all cells examined (Fahey and Sundquist, 1991). In mammalian tissues, the intracellular concentration of GSH (typically 1-10 mM (Griffith and Meister, 1979a; Deneke and Fanburg, 1989) exceeds that of most amino acids and greatly exceeds that of L-cysteine (typically 15-500 FM), (Anderson and Meister, 1987). The high tissue concentrations and wide biological distribu- tion of GSH are consistent with the numerous and varied biological roles played by the tripeptide. Thus, in animals, GSH serves as a storage and transport form of cysteine (Higashi et al., 1977; Tatelshi et al., 1977), as a co- factor in several enzymatic reactions (e.g., glyoxylase I and 11, and formaldehyde dehydrogenase) (Inoue and Kimura, 1995; Koivusalo et al., 1989) and as a protectant against a variety of toxic species (Reed and Fariss, 1984; Smith et al., 1996) (Fig. 1). Among the toxins inactivated by GSH are reactive oxygen species (e.g., hydroxyl radical and hydroperoxides) (Arrick et al., 1982; Izawa et al., 1995), reactive nitrogen species (e.g., peroxynitrite and N203) (Luperchio et al., 1996; Petit et al., 1996; Wink et al., 1996) and covalently reactive electrophiles (e.g., activated metabolites formed by the cytochrome P-450 system and bifuctional DNA alkylating agents used in cancer chemctherapy) (Mannervik and Danielson, 1988; Armstrong, 1997). The finding that elevated GSH levels contribute to the resistance of many tumors to radiation therapy and to chemotherapy using DNA cross-linkmg agents or redox cycling drugs has stimulated interest in the pharmacological control of GSH synthesis as a therapeutic modality (Meister and Griffith, 1979; k i c k andNathan, 1984; Ahmad et al., 1987a; Griffith and Friedman, 1991; Colvin et al., 1993; O’Dwyer et al., 1945; O’Brien and Tew, 1996; Schrode et al., 1996).

The protectant functions of GSH are both important and complex, involving enzymatic as well as nonenzymatic processes. Glutathione reacts directly with some toxic species (e.g., hydroxyl radical and peroxynitrite) (Fig. 1, reaction 8) (Kalyanaraman et al., 1996; Karoui et al., 1996) but requires enzymatic catalysis to detoxify other less reactive species (e.g., GSH peroxidases mediate detoxification of hydroperoxides (Fig. 1, reaction 1 1) (Douglas, 1987). Adduct formation between GSH and some reactive electrophiles (e.g., activated phosphoramide mustard) (Yuan and Smith, 1991) occurs both spontaneously and with GSH S-transferase catalysis (Fig. 1, reaction 13a) (Mannervik and Danielson, 1988; Armstrong, 1997). Glu- tathione also serves to maintain other low-molecular-weight antioxidants, such as ascorbate (vitamin C) and a-tocopherol (vitamin E), in their re-

212 OWEN W. GlUFFITH AND R. TIMOTHY MULCAHY

GLUTAMATE

SH

ELECTROPHILE X

GLUTATHIONE DlSULFlDE

N=O S.

S-NITROSOGLUTATHIONE MERCAPTURIC

ACID

THE ENZYMES OF GLUTATHIONE SYNTHESIS 213

duced, biologically active form (Meister, 1992, 1994). All these protective reactions depend on the cysteine residue of GSH, and the GSH thiol is notable both for its reactivity with toxic species (Jocelyn, 1967) and for its resistance to auto-oxidation (Fahey and Sundquist, 1991; Adang et al., 1988; Griffith and Tate, 1980). Evolution of GSH synthesis may, in fact, have been driven by the need for cells to maintain high intracellular concentrations of cysteine in a form not subject to rapid 02-mediated oxidation (New- ton and Fahey, 1990; Fahey and Sundquist, 1991). Thus, under physiological conditions, the relative rates of trace metal catalyzed autooxidation are cysteine > y-glutamylcysteine > GSH (Fahey and Sundquist, 1991). As a cellular reductant, GSH has the further advantage that glutathione disulfide (GSSG), unlike cystine, is highly soluble at neutral pH.

All the protectant functions of GSH result in its stoichiometric consumption and conversion to nonprotectant products. For example, GSH reacts with most electrophiles by forming a stable thiol adduct (typically a sul- fide); that adduct is then metabolized to a mercapturic acid, which is excreted in the urine (Fig. 1, reactions 13a-13d) (Boyland and Chasseaud, 1996). Although the glutamyl and glycyl residues of GSH are salvaged, the cysteinyl moiety is excreted as part of the mercapturic acid. Because such losses of GSH can be reversed only by de novo synthesis, detoxification of electrophiles can quickly compromise cell survival if cysteine supplies are exhausted or the capacity for GSH synthesis is limited. By contrast, GSH- mediated protection from reactive oxygen species or reactive nitrogen species results mainly in the oxidation of GSH to GSSG (Fig. 1, reactions 7, 8, 11, and 14) (Luperchio et al., 1996; Gilbert, 1990; Zeigler, 1985). In general, GSSG does not accumulate but is rapidly reduced back to GSH by NADPH in a reaction catalyzed by GSSG reductase (also called GSH reductase) (Fig. 1, reaction 12); de novo GSH synthesis is not required (Gilbert, 1990; Zeigler, 1985). If the capacity of GSSG reductase or the sup- - ~

Figure 1. Reactions of GSH synthesis, turnover, and metabolism. Circled numbers corre- spond to the following enzymes or metabolic processes: 1, y-glutamylcysteine synthetase; 2, GSH synthetase; 3a, y-glutamyl transpeptidase (transpeptidation reaction); 3b, y-glutamyl transpeptidase (hydrolysis reaction); 4, y-glutamylcyclotransferase; 5,s-oxoprolinase; 6, cysteinylglycinase or other dipeptidase (both intracellular and extracellular enzymes may be important); 7, nonenzymatic reaction of GSH with NZ03 and other nitrosating species (Luperchio et al., 1996; Petit et al., 1996; Wink te al; 1996); 8, free radical-mediated oxidation of GSH; 9, GSH oxidase; 10, enzymatic and nonenzymatic GSH-dependent transhydrogenations; 1 1, GSH peroxidase; 12, GSSG reductase; 13a, GSH S-transferase; 13b, y-glutamyltranspeptidase; 13c; cysteinylglycinase or other dipeptidase; 13d, cysteine conjugate N-acetyltransferase; 14, spontaneous (Luperchio et a]., 1996). References are provided for enzymes and processes not discussed in the text. Modified from Griffith and Friedman, 1991).

214 OWEN W. GRFFITH AND R. TIMOTHY MULCAHY

ply of NADPH is exceeded, a fraction of the GSSG formed may be prefer- entially excreted from the cell but, even in this situation, all the constituent amino acids are eventually recovered after extracellular degradation of GSSG (McIntyre and Curthoys, 1980).

At physiological levels of NADPH and NADP+, the GSSG reductase reaction is driven strongly in favor of GSH, and the intracellular [GSH]/[GSSG] ratio is typically 2 100 (Gilbert, 1990; Zeigler, 1985). Maintenance of a high [GSH]/[GSSG] ratio maximizes the antioxidant capacity of the GSH pool and minimizes intracellular accumulation of disul- fides (Gilbert, 1990, 1995; Zeigler, 1985). If oxidant stress or other factors cause consumption of GSH to exceed its replenishment through de novo synthesis or GSSG reduction, the consequent shift in the GSWGSSG redox buffer influences a variety of cellular processes. For example, transcription of some genes is regulated by cellular redox status as controlled by the [GSH]/[GSSG] ratio, an effect often mediated through activation of NF-KB or AP-1 (Sen and Packer, 1996). Alterations in GSH synthesis can thus have significant regulatory effects that are just beginning to be elucidated.

This chapter reflects the authors’ interest in mammalian GSH biology and focuses mainly on the enzymology and molecular biology of y-glutamylcysteine synthetase (y-GCS), the first and rate-limiting enzyme of GSH synthesis. Glutathione synthetase, which catalyzes the second step in GSH synthesis will be covered in a subsequent volume. Some of the protectant roles of GSH are also discussed herein as they relate directly to our interest in the pharmacological control of GSH biosynthesis as an approach to cancer chemotherapy. Excellent reviews covering other aspects of GSH biology are available. (Fahey and Sundquist, 1991; Inoue and Kimura, 1995; Mannervik and Danielson, 1988; Armstrong, 1997; Griffith and Friedman, 1991; Colvin et al., 1993; O’Dwyer et al., 1995; O’Brien and Tew, 1996; Schroder et al., 1996; Douglas, 1987; Meister, 1992, 1994; Meister and Anderson, 1983; Akerboom and Sies, 1990).

11. Overview of GSH Biosynthesis

Glutathione is synthesized in both prokaryotes and eukaryotes by the sequential action of y-GCS and GSH synthetase (Fig. 1, reactions 1 and 2, respectively). In mammalian cells, both enzymes are exclusively cytosolic (Griffith and Meister, 1985), and the rate of GSH synthesis is controlled by the amount of y-GCS present, by the availability of L-cysteine (which is often limiting) (Jackson, 1969; Taylor et al., 1996), and by feedback inhibition exerted by GSH on y-GCS (Jackson, 1969; Richman and Meister,

THE ENZYMES OF GLUTATHIONE SYNTHESIS 21s

1975). GSH synthetase apparently has no regulatory role; once synthesized, y-glutamylcysteine is rapidly converted to GSH (Smith et al., 1980). Glu- tathione is not degraded within cells but is instead constantly secreted from cells; intracellular GSH levels are regulated in part by the rate of such transport (Kaplowitz et al., 1996). Despite the constant flow of GSH out of cells, extracellular levels of GSH remain low (1-SO KM) because of the presence of the GSH degrading enzyme y-glutamyl transpeptidase on the external surface of many cells (Griffith and Meister, 1979b). As shown in Figure 1, y-glutamyl transpeptidase catalyzes both the transpeptidation of GSH (reaction 3a) and GSH hydrolysis (reaction 3b). Additional extracellular and intracellular enzymes degrade y-glutamylamino acids and cysteinylglycine, completing the conversion of GSH to its constituent amino acids (Fig. 1, reactions 4-6). Glutamate, cysteine, and glycine, released from GSH, are used for many purposes, including resynthesis of GSH (Fig. 1).

The sequential process of GSH synthesis, secretion, degradation, amino acid uptake, and GSH resynthesis can operate locally (e.g., in the proximal tubule of the kidney) (Griffith, 1981) but is particularly important when it occurs between tissues as an interorgan mechanism of L-cysteine transport (Reed and Fariss, 1984; Taylor et al., 1996; Kaplowitz et al., 1996). Most commonly, the process originates in liver, an organ rich in y-GCS and GSH synthetase that is able to obtain cysteine directly from the diet or from methionine and serine via transsulfuration (Higashi et al., 1977; Tateishi et al., 1977, 1981; Beatty and Reeds, 1980). Glutathione secreted into the plasma by the liver then supports GSH synthesis in extrahepatic tissues that might otherwise be cysteine poor (i.e., they lack the enzymes of transsulfuration and have limited access to dietary cysteine) (Taylor et al., 1996; Kaplowitz et al., 1996). Although intact GSH is not taken up at a significant rate by most peripheral tissues (Abbott et al., 1984), cells expressing y-glutamyl transpeptidase can degrade GSH and take up the resulting cysteinylglycine and cysteine; adjacent cells and cells “downstream” may also benefit by tak- ing up these products. In view of such GSH-dependent interorgan cysteine transport, GSH synthesis in peripheral tissues can be partially dependent on hepatic GSH synthesis.

Similar considerations apply to tumors and may constitute a novel basis for therapy. Thus, many tumors depend on GSH for the detoxification of both chemotherapeutic drugs and reactive oxygen species produced during radiation therapy (Griffith and Friedman, 1991; Colvin et al., 1993; O’Dwyer et al., 1995; O’Brien and Tew, 1996; Schroder et al., 1996). Vis- tica and colleagues (1987) established that some drug-resistant tumor cells express high levels of y-glutamyl transpeptidase on their surface and are

216 OWEN W. GRFFITH AND R. TIMOTHY MULCAHY

therefore able to degrade circulating GSH efficiently, producing abundant L-cysteine for maintenance of tumor GSH levels (Ahmad et al., 1987b). It was suggested that inhibitors of y-glutamyl transpeptidase might be useful in overcoming drug and radiation resistance in such tumors. The absence of potent, specific and nontoxic inhibitors of y-glutamyl transpeptidase pre- vents clinical testing of this hypothesis, but studies with cells and animals clearly indicate that y-glutamyl transpeptidase facilitates intracellular GSH synthesis from extracellular GSH (O’Brien and Tew, 1996; Schroder et al., 1996). It is apparent that a full appreciation of GSH synthesis must take into account not only y-GCS and GSH synthetase, but also the enzymes and transport processes that control substrate availability (reviewed in Bannai and Tateishi, 1986; White et al., 1994).

I11 y-Glutamylcysteine Synthetase

A. PURIFICATION AND PROTEIN CHEMISTRY

y-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) has been purified from Escherichia coli (Watanabe et al., 1986a; Inoue et al., 1993; Huang et al., 1988), Proteus mirabilis (Kumagai et al., 1982), yeast (Dennda and Kula, 1986), Ascaris suum (nematode) reproductive tissue (Hussein and Walter, 1995), Xenopus (toad) liver (Davis et al., 1973), and a wide variety of mammalian sources, including rat kidney (Orlowski and Meister, 1971a; Sekura and Meister, 1977a; Seelig and Meister, 1985a), rat liver (Davis et al., 1973), rat erythrocytes (Seelig and Meister, 1984a, 1985b), hog liver (Mooz andMeister, 1971), sheep erythrocytes (Seelig and Meister, 1985b; Board et al., 1980), bovine lens (Rathbun, 1967a), human erythrocytes (Lebo and Kredich, 1978), and a human malignant astrocy- toma cell line (Srinam and Ali-Osman, 1973). Rat (Huang et al., 1993a) and human (Misra and Griffith, 1998) y-GSC, as well as y-GCS from Try- panosoma brucei, the protozoan responsible for African sleeping sickness (Lueder and Phillips, 1996), have been overexpressed in E. coli and purified to homogeneity. Plant y-GCS has been partially purified from Nicotiana tabacum (tobacco) (Hell and Bergmann, 1990). To date, most enzymologi- cal studies have been carried out with y-GCS isolated from rat kidney, the richest natural source (Orlowski and Meister, 197 1); except where indicated otherwise, the findings and conclusions summarized here are based on studies carried out with that enzyme. Currently, rat kidney y-GCS is most con- veniently isolated using the purification protocol of Seelig and Meister


(1985), which allows preparation of approximately 6 mg of homogeneous enzyme from 60 g of kidneys (25-30 rats).

Although purified mammalian y-GCS is stable indefinitely when stored at -20°C in 25% glycerol (I. Misra and O.W. Griffith, unpublished observations), it is moderately unstable during purification. Such instability probably accounts for the wide variations in specific activity reported for apparently homogeneous enzyme isolated from different mammalian sources (Seelig and Meister, 1985b; Board et al., 1980; Chang and Chang, 1994). The best preparations of y-GCS from rat kidney or erythrocytes have specific activities of approximately 1500 U/mg (1 unit = formation of 1 pmol of producth) (Sekura and Meister, 1977; Seelig and Meister, 1985a,b). This value has also been attained with human y-GCS isolated from E. coli (Misra and Griffith, 1998). Where lower values are found, it is likely that partial de- naturation has occurred during purification. Isolation of enzyme of high specific activity requires the use of fresh tissue or cells, the use of buffers containing L-glutamate and Mg2+ during early purification steps, minimal or no exposure to thiols during purification, and limited duration of exposure to the Mn2+-containing buffer used during ATP affinity chromatography. Mammalian, Candida, and Proteus y-GCS are inactivated by freezing (Kumagai et al., 1982; Dennda and Kula, 1986; Orlowski and Meister, 1971a; Misra and Griffith, 1998), but some reports indicate that the E. coli and Arabidopsis enzymes are stable when stored frozen at -80°C (Watan- abe et al., 1986a; Inoue et al., 1993; Huang et al., 1988; Kumagai et al., 1982; May and Leaver, 1994).

All fully characterized eukaryotic y-GCS are heterodimers composed of a heavy (M, approximately 73,000) and a light (M, approximately 3 1,000) subunit.2 In all cases, the amino acid composition is unremarkable, and there are no established cofactors. There is, however, evidence for bound

* Where enzymes are cloned and expressed without prior isolation of the native enzyme, it is difficult to be certain that all subunits have been identified. For example, a small subunit has not yet been reported for the y-GCS of T. brucei (Lueder and Phillips, 1996; M. Phillips, personal communication), but, as discussed in Section IIIB, the cloned “heavy subunit” of this enzyme contains a 55-amino acid insert that may serve the function(s) of a small subunit. Simi- larly, yeast y G C S (Candida boidinii) is reportedly a homodimer of 60,000-Mr subunits (Dennde and Kula, 1986). but the specific activity achieved was low (0.8 U/mg), and it is not certain that the protein visualized on the SDS-PAGE gels was y-GCS. More recent cDNA sequencing studies with Saccharomyces cerevisiae and Schizosaccharomyces pombe indicate that activity is associated with a protein having high homology to rat y-GCSh (Ohtake and Yabuuchi, 1991; Mutoh et al., 1995). Regulatory subunits in yeast have not yet been identified, but are anticipated.

218 OWEN W. GRlFF’ITH AND R. TIMOTHY MULCAHY

Mg2+ or Mn2+ in the rat kidney and human erythrocyte enzymes (Lebo and Kredich, 1978; Beamer et al., 1980; Chang, 1996). In the absence of thiols, approximately 70% of native rat kidney y-GCS migrates on sodium dode- cry1 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent M, of approximately 100,000 indicating that the subunits are linked by at least one disulfide bond in most of the enzyme (Huang et al., 1993). Reduction of the disulfide bond(s) with P-mercaptoethanol or dithiothreitol allows the heavy and light subunits to be separated on polyacryamide gels (Seelig et al., 1984). Seelig and colleagues showed that the isolated heavy subunit (y-GCSh) was catalytically active and concluded that it contains binding sites for all three substrates and all essential catalytic residues (Seelig and Meister, 1985b; Seelig et al., 1984). The heavy subunit is also subject to feedback inhibition by GSH (Huang et al., 1993; Seelig et al., 1984). Inhibition is competitive with glutamate, suggesting that the GSH and glutamate binding sites are both on the heavy subunit and may be over- lapping (Richman and Meister, 1975) (see also section IIIG).

More recently, Huang and colleagues (1993a) reported that the light subunit (y-GCS serves a regulatory function, modulating the kinetic proper- ties of y-GCSh when the subunits are dimerized to form the holoenzyme. Using recombinant rat liver y-GCS subunits, these investigators reported that K:’” was 18.2 mM for y-GCSh versus 1.4 mM for the holoenzyme (Table 1). The light subunit affected the Ki value for GSH in the opposite di- rection, increasing KGsH from 1.8 mM to 8.2 mM. Since intracellular glutamate concentrations are typically lower than elu for y-GCSh and GSH concentrations are often higher than 1.8 mM, they suggested that the heavy subunit monomer would be essentially nonfunctional under physiological conditions (Huang et al., 1988, 1993a,b). Studies with human y-GCSh show a similar trend with respect to glutamate and GSH, but the changes caused by the absence of the light subunit are much smaller. Thus, KG,‘” increased from 1.9 t 0.2 mM with the holoenzyme to 3.2 2 0.1 mM with y-GCSh and eSH decreased from 3.3 2 0.2 mM to 1 .0 t 0.1 mM (Table 1) (Misra and Griffith, 1998; I. Misra and 0. W. Griffith, unpublished observations). Pre- liminary studies (see Section IIIB) suggest these changes in KG,‘” and eSH, although smaller than those seen with rat y-GCS, may nonetheless be phys- iologically significant for human y-GCS. The question is relevant to cancer biology, as some tumors with elevated GSH levels overexpress only y- GCSh, rather than the holoenzyme (Yao et al., 1995; Mulcahy et al., 1995a). Thus, while it is apparent that overexpression of y-GCSh alone can support higher GSH levels (Bailey et al., 1992; Godwin and Meister, 1992; Mulc- ahy et al., 1994b, 1995), it is possible that maximum rates of GSH synthe-


TABLE 1 Kinetic Parameters for Various y-GCS

Rat Y-GCS".~ 1.4 0.2 0.2 8.2 Rat y-GCShb 18.2 0.2 ND 1.8 Human y-GCS' 1.9 ? 0.2 0.10 2 0.02 0.4 2 0.04 3.3 t 0.2 Human y-GCShC 3.2 IT 0.1 0.13 t 0.01 ND 1.0 2 0.1 Escherichia coli strain Bd 0.5 0.09 0.1 -2

E. coli strain KM y-GCS" 1.7 0.2 ND -4 E. coli strain W y-GCS" 0.7 0.1 ND ND

Proteus mirabilis y-GCS' 1.6 0.15 0.16 ND Candida boidinii y-GCS' 1.4 0.4 0.2 3.1 Typanosoma bruceiy-GCSE.h 0.24 2 0.02 0.69 t 0.13 0.71 2 0.01 1.1 2 0.2 Ascaris suum' 0.94 2 0.05 0.41 1.41 2 0.01 0.11 2 0.01 Nicotiana tabacum y-GCY 10.4 2 0.6 0.19 2 0.05 ND 0.42 5 0.12

ND, not determined.

Huang et al., 1998; Huang et al., 1993; Misra and Grifith, 1998; Watanabe et al., 1986;' Kumagai et al., 1982;fDennda and Kula, 1986; The enzyme (M, = 77,400) was cloned and expressed in E. coli. It is unclear whether T. brucei contains a light subunit that would modify these kinetic parameters. As Lueder and Phillips (1996) point out, there is no need for a y- GCSl tabring e'" into the physiological range; Lueder and Phillips, 1996; Hussein and Wal- ter, 1995;' Hell and Bergmann, 1990.

sis are compromised by the absence of y-GCS1 (see Section I11 B4). Several studies suggest that the survival of cancer cells exposed to drugs detoxified by GSH (e.g., bifunctional DNA alkylators) depends less on the initial concentration of GSH in the cells and more on their ability to synthesize additional GSH rapidly, once their initial supply is consumed (Lee et al., 1989; Moore et al., 1989; Meister, 1991).

Although E. coli and P. mirabilis y-GCS are monomers (M, approximately 60,000), their kinetic parameters are not greatly different than those of the mammalian enzyme (Table 1); amino acid binding and inhibition by GSH are very similar to rat y-GCS (Huang et al., 1988). E. coli y-GCS does differ from the mammalian enzyme in sensitivity to some active site-directed inhibitors as discussed later. Kinetic constants for y-GCS from a yeast (Dennda and Kula, 1986), protozoan (T. brucei) (Lueder and Phillips, 1996), helminth (A. suum) (Hussein and Walter, 1995), and higher plant (N. tabacum) (Hell and Bergmann, 1990) are also given in Table 1 . Most of the values reported are similar to those obtained with rat kidney y-GCS, but some differences may be biologically significant. The y-GCS isolated from T. brucei exhibits a relatively low Ki value for GSH (Lueder and Phillips, 1996), but this organism converts most of its GSH to trypanothione (i.e., N',


I@-bis(glutathiony1)spennidine) and only 10-27% of the total GSH pool is present as GSH (concentration < 1 mlM) (Henderson et al., 1987; Fairlamb and Cerami, 1992). The relatively low KGsH may thus serve to keep GSH synthesis and GSH conversion to trypanothione in balance without significantly limiting the overall pathway. The significance of the relatively high Fmys and low el" for T. brucei y-GCS remains unclear in the absence of data on cellular amino acid levels. Assuming that the cysteine level is not unusually high and that the kinetic values are not modified by an as yet unidentified small subunit,2 the high Fmys would be expected to limit the maximum rate of GSH synthesis.

Consistent with its distant evolutionary relationship to mammalian y- GCS isoforms (Newton and Fahey, 1990; Fahey and Sundquist, 1991), (section IIIB), tobacco (N. tabacum) y-GCS shows the greatest divergence in its kinetic constants; C''' is markedly higher and eSH is lower than the corresponding values in bacteria or animals (Hell and Bergmann, 1990). Be- cause the values reported are similar to those seen with rat y-GCSh, one must consider the possibility that a small subunit was lost in purifying the plant y-GCS. There is no evidence that this occurred, and the tobacco y- GCS isolated is apparently a homodimer of M, 34,000 subunits (Hell and Bergmann, 1990), inconsistent with a mammalian-type quartemary structure. The kinetic constants for N. tubacum do suggest that GSH synthesis is likely to be limited by glutamate availability and to be significantly inhibited by endogenous GSH (reportedly 1-6 mM in plants) (Hell and Bergmann, 1990; Klapheck et al., 1987).

B. CLONING, SEQUENCING, AND EXPRESSION OF Y-GCS SUBUNITS

Watanabe et al. (1986b) cloned and sequenced the genomic DNA encoding E. coli y-GCS, ushering in the molecular era in GSH investigations. Since that time, y-GCS cDNAs have been cloned and sequenced from several species, including rat (Yan and Meister, 1990), mouse (Reid et al., 1997a,b), human (Gipp et al., 1993), yeast (Ohtake and Yabuuchi, 1991; Lisowsky, 1993; Coblenz and Wolf, 1995; Mutoh et al., 1991), higher plants Arubidopsis thaliana3 (May and Leaver, 1994), protozoa (7'. brucei (Lueder

A partial sequence (651 bp) showing 91% nucleotide identity and 96% amino acid identity to the A. fhaliana sequence has been reported for Brussica juncea, a heavy metal accumu- lator from the mustard family (Schafer et al., 1997). In this species, GSH is polymerized into heavy metal binding phytochelatins, which play a role similar to that of metallothioneins.


and Phillips, 1996),) L. tarentolae (Grondin et al., 1997), and other bacteria (Powles et al., 1996). The genomic sequences for the heavy and light subunit of human y-GCS, including the 5’-flanking region of the genes, have also been reported (Mulcahy and Gipp, 1995; Mulcahy et al., 1997). A sum- mary of the sequence data, including accession numbers for those sequences available from DNA databases, is compiled in Table 2.

1. Eukaryotic y-GCS Catalytic Subunit

There is a variable degree of sequence identity among the cDNA sequences for the eukaryotic y-GCS catalytic (heavy) subunit (y-GCSh), with the highest degree of similarity (90-95%) among the cDNAs of mammalian origin. There is also a moderate degree of homology between the mammalian and yeast cDNA sequences. By contrast, a much lower degree of sequence similarity exists between these cDNAs and those encoding the E. coli or Arabidopsis proteins.

As expected, a comparable relationship is observed for the deduced amino acid sequences of the various y-GCS proteins. As shown in Table 2, the bacterial and Arabidopsis y-GCS cDNAs encode catalytic proteins that are smaller than those of the other species examined; the bacterial and plant proteins share only limited amino acid sequence identity (<lo% and <20%, respectively) and modest similarity (29-33% and 16%, respectively) with mammalian y-GCSh. By contrast, the proteins translated from both the T. brucei and the L. torentolae cDNA sequences have considerable sequence similarity with the mammalian proteins (approximately 43% and approximately 45%, respectively), although both protozoan proteins contain an unique 55-amino acid insert absent in the other species. Based on a comparison of the primary structure of the deduced y-GCS proteins for these various species, May and colleagues have suggested that the bacterial and Arabidopsis proteins may have evolved independently from the rest (May and Leaver, 1994). The calculated evolutionary relationship among the various y-GCS catalytic subunit proteins based on their deduced primary structure is summarized in the phylogenic tree shown in Figure 2. By this analysis, the ancestoral y-GCS hypothesized by Newton and Fahey (1990) and by Fahey and Sundquist (1991) would have evolved in one line leading to purple bacteria (E. coli and Thiobacillus), protozoa and animals, and in another line leading to cyanobacteria and plants. It would therefore be of considerable interest to have sequence data for a y G C S from cyanobacteria to determine its relationship to Arabidopsis.

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As shown in Figure 2, the protozoal, yeast and animal y-GCS sequences are more closely related to one another than to the bacterial and Arabidop- sis sequences. When the latter sequences were excluded from the comparison, 145 invariant amino acids were identified in the sequences for the re- maining seven y-GCS proteins. These residues are not randomly distributed throughout the proteins but are located mainly within three relatively large highly conserved regions and in several smaller stretches of homology. Many of the homologous sequences contain conserved lysine and arginine residues, amino acids that have been implicated in specific interactions with GSH in other GSH-binding proteins (Douglas, 1987, Stole and Meister, 1991).

As discussed in section IIIF, studies with mammalian y-GCS indicate the presence of a conserved cysteine residue in the glutamate binding site; reaction of that residue with cystamine, various chloroketones, y-methylene- D-glutamate, or nitric oxide donors blocks enzymatic activity. Although studies definitively identifying a specific residue have not been reported, a single conserved cysteine is found in one of the highly conserved regions in six of the seven related y-GCS catalytic subunit proteins (Fig. 3). The conserved cysteine is located within a glycine-rich region having the conserved motif M(A/G)FGMGXXCLQ (Lueder and Phillips, 1996). It has been suggested that this sequence may function like the glycine-rich P-loop common to phosphate binding sites to position the y-phosphate of ATP close to the glutamate binding site, thereby facilitating formation of y-glutamyl-phosphate, the enzyme-bound reaction intermediate (see Section IIIF) (Lueder and Phillips, 1996). However, undisputed identification of a specific role for any amino acid residue of y-GCS requires additional experimentation. Such

yGCS (catalytic)

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Figure 2. acid sequences deduced from reported cDNA sequences (Table 2).

Phylogenetic relationship of yGCS catalytic subunits based on alignment of amino

224 OWEN W. GIUF'FITH AND R. TIMOTHY MULCAHY

t Figure 3. Region of high amino acid sequence identity in the y-GCSh proteins from seven species. Boxes, identities; arrow, presence of a conserved cysteine residue in six of the seven proteins. Numbers to the right, amino acid residue position.

studies should be greatly facilitated by the current availability of cDNAs that can serve as targets for site-directed mutational strategies.

2. y-GCS Regulatory Subunit

Although the catalytic function of the heavy subunit was readily established, the function of the light subunit (y-GCS1) remained a mystery until Huang et al. (1993a,b) demonstrated that it regulated the affinity of the catalytic unit for its substrates and inhibitors, as noted previously. To date, cloning of y-GCS light subunit cDNAs has been reported only for rat (Huang et al., 1993a), mouse (Reid et al., 1997b; Mulcahy et al., 1997), and human (Gipp et al., 1995). Considering the sequence similarity among the y-GCS heavy subunits it might be assumed that a dimeric quaternary structure is characteristic of the y-GCS in other species as well. However, at least in the case of A. thaliana and T. brucei, all available biochemical evidence suggests that y-GCS is monomeric and functional in the absence of a specific regulatory subunit (Lueder and Phillips, 1996; May and Leaver, 1994). For T. brucei (and, by analogy, L. torentolae), it is possible that the 55-amino acid insert present in these enzymes but missing from the mammalian y-GCSh serves the functions of a y-GCS1 (Lueder and Phillips, 1996). The insert does not, however, show sequence similarity to y-GCS1 (M. Phillips, personal communication). The rat, murine, and human light subunit cDNAs each encode proteins 274 amino acids in length, having deduced molecular weights of approximately 3 1 kDa. Human y- GCS, shares 96% amino acid sequence homology with its rat and mouse counterparts.


3. y-GCSh and y-GCS, mRNA Expression

Hybridization of Northern blots with radiolabeled probes corresponding to rat or human y-GCSh cDNAs identifies a 4.1-kb transcript in total RNA isolated from rat tissues, while two transcripts (3.2 kb and 4.1 kb) are evident in RNA isolated from tissues of human origin (Gipp et al., 1992, 1995) (Fig. 4). Two transcripts are also identified in RNA hybridized with radiolabeled y-GCS1 cDNA probes in each species; 1.8- and 5.2-kb transcripts

GCS Heavy GCS Light Subunit Subunit

Spleen

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Testes

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Sm. Int.

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PBL

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Heart

Brain

Placenta

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Liver

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Figure 4. Northern blot analysis of mRNA isolated from various human normal tissues. Blots were hybridized with a radiolabeled 736-bp probe from the human y-GCS1 cDNA (top) or a radiolabeled 764-bp probe from the human y-GCSh cDNA (bortom). PBL, peripheral blood leukocytes. From Mulcahy et al. (1997).


are detected in rat tissues (Huang et a]., 1993a) and 1.4- and 4.1-kb transcripts are detected in human tissues (Gipp et al., 1995) (Fig. 4). It should be noted that, in most tissues, the larger of the y-GCS1 transcripts is present in low abundance and is not always detectable by Northern blot analysis using total RNA. The reason for the difference in light subunit transcript size and the functional significance of such difference, if any, remains to be determined.

Like y-GCS enzymatic activity itself, steady state levels of mRNA for the mammalian y-GCSh and y-GCS I subunits are highly tissue dependent (Fig. 4). Interestingly, y-GCSh and y-GCSI subunit mRNA expression is not well correlated in any individual tissue examined to date. For example, y-GCS1 mRNA expression is highest in human skeletal muscle where y- GCSh mRNA expression is relatively low. An opposite relationship is found in lung. It is anticipated that a better understanding of tissue-specific transcriptional control of y-GCS subunit expression will help elucidate the well- documented tissue-dependent pattern of y-GCS enzymatic activity.

Disparate expression of the two y-GCS subunit mRNAs is also evident in total RNA isolated from drug-resistant tumor cells expressing elevated y- GCS activity and increased levels of intracellular GSH. Tumor cells expressing this resistance phenotype consistently overexpress y-GCSh transcripts, whereas increases in regulatory subunit transcripts are observed infrequently (see Section IIID).

4. Expression of Recombinant y-GCSh and y-GCSl Proteins in Mammalian Cells

The cDNAs encoding the light and heavy subunits of human y-GCS have been cloned into mammalian expression vectors and transfected into COS- 7 cells at various molar ratios (the total amount of DNA was held constant) (Mulcahy et al., 1995). Introduction of the light subunit cDNA alone did not significantly alter y-GCS activity in homogenates, but transfection with the catalytic subunit cDNA increased y-GCS activity in direct proportion to the quantity of y-GCSh cDNA used to transfect the cells (Fig. 5A). It should be noted that the high-performance liquid chromatography (HPLC)-based assay used to monitor y-GCS activity employed a high glutamate concentration in order to determine activity of y-GCSh accurately, even when it is not complexed to y-GCSI. The y-GCS activity detected was thus proportional to the amount of y-GCS catalytic subunit present in cellular lysates and was not necessarily indicative of the presence of functional holoenzyme.


160

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Figure 5. (A) y-GCS activity measured in transfectants 48 h after transfection. COS cells were transfected with the two human y-GCS subunit cDNAs alone or in combination at various molar ratios as shown in parenthesis. The results shown are means and standard errors of 4 4 individual transfections. From Mulcahy et al. (1995). (B) GSH levels measured in COS cells 48 h after transfection. COS cells were transfected with the human y-GCS subunit cDNAs as described in A.

Glutathione levels in transfectants overexpressing the y-GCS cDNA were not significantly changed in comparison to controls, although a modest, but consistent, elevation was detected (Fig. 5B). Transfection with the heavy subunit cDNA alone resulted in an elevation of GSH (approximately 1.6-fold). However, in cells transfected with y-GCSh cDNA in combination with y-GCS1 cDNA in any molar ratio, GSH elevations were maximal, approaching 300% of control. The presence of the light subunit apparently greatly enhances the in vivo catalytic efficiency of the exogenous catalytic subunit. For example, although the relative y-GCS activity in extracts pre- pared from cells transfected with y-GCS1 and y-GCSh in a 4: 1 molar ratio was only about 20% of that in cells transfected with an equal total amount of y-GCSh cDNA alone, the GSH concentration was significantly higher in


the former group (160 vs. 114 nmoymg protein). The results of these transfection studies are consistent with the kinetic analysis reported by Huang et al. (1993a,b) and support the postulated significance of the light subunit in y-GCS regulation in vivo.

5. Expression of Recombinant y-GCSh and y-GCSI in E. coli

Huang et al. (1993a,b) have reported the expression of rat kidney y- GCSh in E. coli BL21 (DE3). The enzyme was purified to apparent homogeneity by a combination of ion-exchange (DEAE-Sephacel) and affinity chromatography (ATP-agarose); 3.7 mg of y-GCSh was obtained from 9.8 g of packed cells. Sequence analysis indicated that the N-terminal methionine residue was missing, but the expressed catalytic subunit exhibited the expected molecular weight on SDS-PAGE, and its was similar to that determined for y-GCSh isolated from native rat kidney enzyme. The specific activity was 385 U/mg in the standard activity assay containing 10 mM L-glutamate but was 1030 U/mg when assayed in the presence of 50 mM L- glutamate, a result consistent with the high KZlu observed in the absence of the light subunit.

Huang et al. (1993a) have also expressed rat y-GCS holoenzyme in E. coli BL21 (DE3); the cDNAs for the heavy and light subunits were incorporated into an expression vector (pT7-7), which contains two T7 promot- ers in opposite directions, immediately followed by the heavy and light subunit constructs. Recombinant y-GCS was purified similarly to recombinant rat y-GCSh, but a final HPLC gel-filtration step was added. The final product (3.4 mg from 10.8-g cells) had a specific activity of 1250 U/mg and was homogeneous by SDS-PAGE; kinetic constants for substrates and inhibition by GSH were consistent with the native rat kidney enzyme.

I. Misra and 0. W. Griffith recently expressed human y-GCSh (unpublished observations) and y-GCS holoenzyme (1998) in E. coli BL21(DE3) using cDNA obtained from Mulcahy and colleagues (Mulcahy et al., 1995; Gipp et al., 1992, 1995). Expression of y-GCSh was accomplished without difficulty, but attempted co-expression of y-GCSh and y-GCS, from a single plasmid did not produce acceptable yields of the holoenzyme. For this reason, cotransfection with separately selectable plasmids bearing y-GCSh and y-GCS1 constructs was carried out; expression of approximately 32 mg y-GCS in 100-g cells was typically obtained. The enzymes were purified essentially as described previously for the native rat kidney enzyme (ammo- nium sulfate fractionation, Q-Sepharose chromatography, ATP affinity


chromatography), except that gel filtration chromatography and anti-E. coli y-GCS immunoaffinity chromatography were carried out before and after ATP affinity chromatography, respectively, to remove residual E. coli y- GCS. Kinetic constants for recombinant human y-GCSh and yGCS holoenzyme are given in Table 1; final specific activities were about 1400 U/mg and about 1500 U/mg, respectively.

C. CLONING OF -y-GCS GENES AND REGULATION OF EXPRESSION

1. y-GSHI and Gcsl: The Yeast y-GCS Genes

The GSHl gene, encoding Saccharomyces cerevisiae y-GCS, was cloned by complementation of a y-GCS deficient mutant by Ohtake and Yabuuchi (1991). The gene was isolated as a 4.4-kb Sphl fragment that was capable of restoring y-GCS activity, GSH synthesis, and cell proliferation, when introduced into the mutant cells. The clone included approximately 1.3 kb of 5'-untranslated flanking sequence containing multiple TATA and CAAT boxes upstream of the ATG initiation codon. The GSHl gene was subsequently mapped to chromosome X by Southern analysis of S. cerevisiae chromosomal DNA fragments (Ohtake and Yabuuchi, 1991).

A similar strategy was employed by Coblenz and Wolf (1995) to clone Gcsl, the y-GCS homologue in the fission yeast, Schizosaccharomyces pombe. These investigators identified a 13-kb DNA fragment that comple- mented cadmium sensitivity in GSH-deficient mutants; a 3463-bp fragment encoding y-GCS was isolated. In contrast to the S. cerevisiae GSHI gene, which is continuous, the Gcsl gene reportedly contains two introns. Two putative TATA and CAAT sequences were identified in the 5'-flanlung region of the Gcsl gene. The two yeast y-GCS homologues share 41% identity and 74% similarity at the amino acid level.

Although the cis-acting elements active in the regulation of the expression of the yeast y-GCS genes have not been well characterized, recent studies have shown that a YAP-1 responsive element (YRE) present in the promoter of the GSHI gene of S. cerevisiae is involved in transcription of the gene in response to exposure to cadmium (Wu and Moye-Rowley, 1994). The S. cerevisiae YAP- 1 is a homologue of the mammalian AP- 1 protein and has been shown to play a prominent role in adaptive responses to drug chal- lenge. The YRE in the GSHl gene is located 380 bp upstream of the start codon and is identical to the the AP- 1 responsive element present in simian virus 40 (SV 40) promoter in 11 of 12 base positions (Wu and Moye-Row- ley, 1994). The YRE in the S. cerevisiae gene binds YAP-1 protein in vitro,


and mutagenesis of the YAP-1 consensus sequence inhibits binding of YAP- 1 protein, rendering the GSHl promoter nonresponsive to exogenously expressed YAP-1. Stephen et al. (1994) recently extended these observations, demonstrating that basal expression of the GSHl gene, as well as gene in- duction after exposure to hydrogen peroxide were both YAP-1 dependent. Collectively, the available data indicate that YAP-1 directs expression of S. cerevisiae y-GCS and contributes to the adaptive response to specific toxic insults.

2. The Human Catalytic Subunit (y-GCSh) Gene

The human y-GCSh gene has been isolated and cloned from a human foreskin fibroblast P1 library (Mulcahy et al., 1997). Southern analysis of an approximately 100-kb fragment isolated from the P1 clone suggests that this fragment encompasses the entire y-GCSh gene. Although determination of the entire introdexon structure of the gene has not been completed, preliminary studies of the 5’ end of the gene indicate that the first few exons are separated by large intronic stretches (R.T. Mulcahy, unpublished observations). The human and mouse genes have been mapped to chromosome band 6p12 and to chromosome 9, band D-E, respectively (Tsuchiya et al., 1995; Walsh et al., 1996; Sierra-Rivera et al., 1995, 1996; Jaiswal, 1994).

A 4.2-kb fragment of the human y-GCSh genomic clone, spanning the sequence -3802 to +465 of the gene, has been cloned and sequenced (Mul- cahy and Gipp, 1995; Mulcahy et al., 1997). The first approximately 200 bp upstream of the ATG start codon are highly GC-rich (about 75%). Sequence analysis identified several consensus transcription factor binding sites, including AP-1, Sp-1, AP-2, and NF-KB motifs. The 5‘-flanking sequence contains a canonical TATA and CAAT box combination, but transcription of the human y-GCSh subunit gene is complex and involves transcription from multiple distinct initiation sites, apparently using TATA-dependent and -independent mechanisms (Mulcahy and Gipp, 1995). The high GC con- tent, utilization of multiple initiation start sites, and the presence of multiple Sp-1 binding sites in the gene are features common to “housekeeping genes”; these findings are therefore in keeping with the ubiquitous expression of the y-GCSh gene in virtually all cells types. Although several transcription initiation sites were identified in each of several cell lines examined, others were only evident in specific cells; such diversity may be due to cell- or tissue-type specific initiation site utilization. This possibility is currently under investigation, as is also the possibility that differential site utilization occurs when gene expression is induced by xenobiotics.


A GAG trinucleotide repeat polymorphism was recently identified in the 5’-untranslated region of the human y-GCSh gene (Walsh et al., 1996). Ge- netic analysis of 50 unrelated Caucasians identified three alleles as follows: A1 (9 repeats, frequency = 35%), A2 (8 repeats, frequency = 11%), and A3 (7 repeats, frequency = 54%). Although certain trinucleotide repeats have been associated with recombinatory events, the functional significance of this particular allelic polymorphism, if any, is unknown.

Identification of cis-acting elements directing constitutive and P-naphthoflavone (P-NF) induced expression of the human y-GCSh gene has recently been reported (Mulcahy et al., 1997). A series of deletion mutants created from the 5’-flanking region of the gene were cloned into a luciferase reporter vector and transfected into human hepatoblastoma HepG2 cells (Figure 6). Maximal constitutive expression was directed by sequences between -202 and +22, and by a distal fragment spanning the region -2752 to -3802. The former sequence includes the consensus TATA box. Expo- sure to a concentration of P-NF, which induces an increase in the steady- state levels of the endogenous y-GCSh and y-GCS1 mRNA levels, increased luciferase only in HepG2 cells tranfected with promoterheporter fusion genes containing the full-length - 3802: t465 fragment. Hence, elements directing both the constitutive and P-NF-induced y-GCSh subunit gene are present in the distal portion of the 5’-flanking sequence.

The distal portion of the y-GCSh promoter contains two consensus antioxidant responsive elements (AREs) separated by 33 bp. Since AREs have been shown to direct both constitutive and P-NF-induced expression of several genes (Jaiswal, 1994), the role of these two AREs (designated ARE3 and ARE4) in y-GCSh expression was examined. Enhancer and mutational analyses showed that ARE4, the more distal of the pair, was required for both constitutive and P-NF-induced expression. The data also suggest that the presence of ARE3 may modulate both constitutive and induced expression. Since ARE4 also contains an embedded consensus AP- 1 motif, additional mutational analyses are required before definitive identification of which of these potential cis-acting elements mediates these responses can be completed.

3. The Human Regulatory Subunit (y-GCSl) Gene

The gene encoding the human regulatory subunit was also recently cloned from a normal human foreskin fibroblast PI library (R.T. Mulcahy, unpublished observations) and localized to chromosome lp21 (Tsuchiya et al., 1995; Sierra-Rivera et al., 1995). Like the catalytic subunit gene, the 5’-


i


flanking region of the y-GCSI gene is highly GC-rich and contains motifs for several common transcription factors, including AP- I and Sp- 1. Primer extension and S 1 nuclease protection analysis demonstrated that multiple initiation sites are used when the gene is transcribed; some initiation sites are proximal to putative TATA sequences, while others are apparently TATA independent. As is true of the GCSh gene, the significance of these multiple start sites and whether they are used differentially in various tissues or in response to specific inducing events remains to be determined.

Functional analysis employing promoter deletion mutants cloned into luciferase reporter vectors identified a consensus ARE sequence in the proximal 5‘-flanking region of the gene that functions as a genuine ARE when cloned into an enhancerheporter vector; both basal expression and expression in response to (3-NF were increased. However, although mutation of this ARE sequence in the GCSh genomic DNA reduced constitutive and p- NF-induced expression, mutation of the sequence failed to eliminate the p- NF responsiveness. Therefore, regulation of the human y-GCS1 gene after p-NF exposure appears to be distinct from that of the catalytic subunit gene.

D. ROLE OF y-GCS OVEREXPRESSION IN RESISTANCE TO CANCER

Resistance to the cytotoxic effects of common chemotherapeutic agents represents a serious problem limiting the effectiveness of conventional chemotherapy. Resistance mechanisms can be multifactorial, particularly late in the evolutionary history of the resistant phenotype, but one of the ear- liest and most common biochemical changes detected in preclinical models of alkylating agent resistance is an elevation of intracellular levels of GSH (Griffith and Friedman, 1991; Colvin et al., 1993; O’Dwyer et al., 1995; O’Brien and Tew, 1996; Schroder et al., 1996). The relevance of GSH ele-

CHEMOTHERAPY

4 Figure 6. Mutational analysis of the y-GCSh 5‘-flanking sequence. A 4.2-kb HindIII fragment (-3802:+465) from the 5’4anking region of the y-GCSh gene was cloned into the luciferase reporter vector, pGL3-basic. to create the fusion gene -3802/GCShS’-luc. Progres- sively smaller transgenes were created by further digestion of this fusion gene with the various restriction enzymes shown at the map at the bottom. The resultant 5’-deletion mutants were transfected into HepG2 cells and used to locate cis-acting elements involved in the constitutive and P-NF-induced expression of the GCSh gene. This analysis, combined with site-directed mutagenesis (T to G in ARM) showed that a distal antioxidant-responsive element (W) was required for both constitutive and induced expression of the gene. From Mulcahy et al. (1997).

234 OWEN W. GRIFF’ITH AND R. TIMOTHY MULCAHY

vations to drug resistance has been extended to human malignancies by recent studies reporting increased GSH levels in cells isolated from tumors after treatment failure or from patients who had received prior chemotherapy (Lewis et al., 1988; DeVries et al., 1989; Choy et al., 1991). The linkage between GSH and drug resistance is further strengthened by studies indicating that pharmacological depletion of intracellular GSH results in drug sensiti- zation (Griffith and Friedman, 1991; Colvin et al., 1993; O’Dwyer et al., 1995; O’Brien and Tew, 1996; Schroder et al., 1996) and by the recent demonstration that cotransfection of the two y-GCS subunit cDNAs results in increased y-GCS activity, GSH levels, and resistance to melphalan (L-

PAM) (Mulcahy et a]., 1995). The mechanisms by which GSH protects cancer cells are presumably

similar to those applicable to normal cells (see Section I). Consistent with this view, both elevated levels of GSH S-transferase isozymes and elevated GSH levels are frequently seen in tumors resistant to various chemotherapeutic agents; the enzyme and the co-substrate required for drug-GSH conjugate formation are thus both overexpressed. In addition, GSH has a functional role with certain DNA repair enzymes (Colvin et al., 1993; O’Dwyer et al., 1995; O’Brien and Tew, 1996), and its elevation can thus facilitate repair of drug-induced DNA lesions. More recently, energy-dependent drug transport systems capable of recognizing and transporting drug-GSH conjugates out of cells have been identified, providing yet another mechanism by which increased levels of intracellular GSH may contribute to protection against cytotoxic chemotherapy drugs (Awashti et al., 1994; Ishikawa and Ali-Osman, 1993).

Mulcahy and others anticipated that the GSH elevations commonly associated with resistance would reflect stable changes in the regulation of GSH biosynthesis, specifically in expression of one or both of the GSH biosynthetic enzymes. Although there is no precedent for increased levels of GSH synthetase, there is ample precedent in the toxicology literature for increased y-GCS activity developing in response to exposure to toxic agents. For this reason, the contribution of y-GCS gene expression to the drug resistant phenotype has been most thoroughly studied. Several dmg-resistant cell lines were found to exhibit alterations in GSH homeostasis characterized by elevations in intracellular GSH levels, increased y-GCS activity, and increased steady-state levels of the y-GCS heavy subunit mRNA (Godwin et al., 1992; Mulcahy et al., 1994b, 1995) (Fig. 7). Although far less common, steady-state levels of the light subunit mRNA are also be elevated in some drug resistant populations (R.T. Mulcahy, unpublished observations). Subsequent studies established that the increase in y-GCSh


DU-145

< 28s Figure 7 Increased steady-state y-GCSh mRNA lek - els in drug resistant DU-145 prostate tumor cells RNA isolated from a senes of drug-resistant DU-145 prostate carcinoma cells was size fractionated in 1.2%

< 18s formaldehyde/agarose gels and then transferred to a 01-

trocellulose membrane. Increased levels of y-GCS,,- specific transcripts were identified RNA isolated from the drug-resistant lines (DU-145M6, IM5 , and /M4 5 ) The lane labeled “OUT” contains RNA isolated from resistant DU-14.5M.5 cells maintained in the absence of drug for a 2-year penod.

message level is secondary to an up-regulation of transcription of the y-GCS catalytic subunit gene (Yao et al., 1995; Mulcahy et al., 1994). The precise mechanisms responsible for the increased rate of transcription have not been fully elucidated but, in the case of a cisplatin-resistant ovarian carcinoma line, the increased expression of the GCSh gene is apparently the result of constitutive overexpression of AP-1 activity (Yao et al., 1995). This mechanism is apparently not responsible for the increased transcription rate detected in a similar series of prostate cancer cells made resistant to L-PAM (A. Wild and R.T. Mulcahy, unpublished observations). Additional studies are necessary to elucidate the full range of possible activation mechanisms.

In another recent advance, y-GCS and the multidrug resistance protein (MRP) have been shown to be coordinately overexpressed in cisplatin-resistant HL-60 human leukemia cells (Ishikawa et al., 1996). Similarly, a strong correlation between y-GCS and MRP expression in biopsies of human colon carcinomas has also been reported (Kuo et al., 1996). Since MRP is an ATP-dependent efflux pump capable of trafficking multivalent anions. including GS-conjugates, from cells (Ishikawa, 1992), it may constitute an important element in a multicomponent GSH-dependent resistance pathway which, in addition to MRP, includes y-GCS and perhaps various GSH S - transferases. The data suggesting that the expression of the MRP/GS-conjugate pump are closely linked with y-GCS expression have prompted spec- ulation that the expression of genes encoding the various member protein\ are coordinately regulated in resistant cells in ways that remain undefined. Therapies designed to overcome these resistance mechanisms are being dc- veloped (see Section I11 G3).

236 OWEN W. GRIFFITH AND R . TIMOTHY MULCAHY

E. POSTTRANSLATIONAL REGULATION

The possibility that y-GCS expression is also regulated at the posttranslational level has recently been suggested. Lu et al. (1988) reported that hepatic GSH synthesis is down-regulated in response to hormones known to mediate their effects through the activation of distinct signal transduction pathways. Using a series of inhibitors of specific signaling pathways, these investigators determined that the hormone-specific inhibition of GSH synthesis was mediated by the Ca2+-dependent, protein kinase C (PKC), and protein kinase A (PKA) pathways. They further demonstrated that the in- hibitory effect on GSH synthesis was secondary to y-GCS inhibition and speculated that the underlying mechanism(s) might include direct phosphorylation of the enzyme itself (Sun et al., 1996). Subsequent studies have shown that PKC, PKA, and Ca2+/calmodulin-dependent kinase I1 are all capable of phosphorylating purified rat kidney y-GCSh on serine and threonine residues in a Mg2+ concentration-dependent fashion. Furthermore, inhibition of y-GCS activity was correlated with the degree of phosphorylation. Phosphorylation of y-GCSh was also detected in rat hepatocytes treated with dibutryl cyclic adenosine monophosphate (CAMP), providing evidence for a role of phosphorylatioddephosphorylation events in the regulation of y-GCS activity in vivo (Sun et al., 1996).

Another potential posttranslational control mechanism is suggested by studies reported by Ochi (1 993, who examined the mechanism for the increased y-GCS activity induced in V79 cells by exposure to hydrogen peroxide. In contrast to the response observed in H202-treated yeast, increased y-GCS activity in V79 cells was not due to up-regulation of y-GCSh gene transcription. Furthermore, since the H202-induced increase in activity did not involve the generation of reactive radicals and could be reversed by chemical reduction (e.g., with dithiothreitol or GSH), Ochi suggests a mechanism involving direct oxidation of yGCS itself (Ochi, 1995). Studies with purified enzyme are necessary to confirm these observations and elucidate the molecular basis for the findings.

F. CATALYTIC MECHANISM

1. Substrate Specificity

The substrate specificity of rat kidney y-GCS has been extensively in- vestigated by Meister and coworkers. (Orlowski and Meister, 1971a; Sekura and Meister, 1977; Sekura et al., 1976; Griffith and Meister, 1977) (Table 3).

THE ENZYMES OF GLUTATHIONE SYNTHESIS

TABLE 3 Substrate Specificity of Rat ffidney y-GCS

237

~

Relative Activity Relative Activity as Substrate" as Substrateb

Glutamate Analogue (%I Cysteine Analogue (%)

L-Glutamate 100 L-Cysteine 96 L- Aspartate <0.5 L-Homocysteine 25 L-a- Aminoadipate C0.5 S-Methyl-L-cysteine 85 D-Glutamate 1.2' DL-Allylglycine 74 Glutarate <0.5 P-Chloro-L-alanine 59 y- Aminobutyrate <0.5 Glycine 3.7 DL-a- Aminometh ylglutarate 5.9 L- Alanine 10 DL-a-hnomethylsuccinate 2.5' L-a-Aminobutyrate 100

DL-P- Aminoadipate 5.1 L-Norleucine 3.4 N-Methyl-L-glutamate 23.5 L-a- Aminohepanoate 1 .o a-Methyl-DL-glutamate 5.9 L-Threonine 20

threP-P-Hydroxy-oL-glutamate 11.0 DL-Homosenne 5.5 y -Methyl-DL-glutamate c0.5 DL-P-Amino-iso-buytrated 33-63 fhreo-y-Hydroxy-L-glutamate 4.4

P-Glutamate 17.6 L-Norvaline 48

P-Methy 1-DL-glutamate 1.6 L-Swine 3.1

e~rhro-y-Hydroxy -L-glutamate 4.0

Rat kidney y-GCS was assayed in the presence of 10 mM glutamate analogue (20 mM for D L - ~ ~ x -

tures), 10 mM L-a-aminobutyrate and 20 mM MgClz; Pi formation was monitored (Sekura and Meister, 1977a). Peptide products were confirmed for all analogues showing >4% the activity with L-glutamate.

and 20 mM MgCIZ; Pi formation was monitored (Ochi, 1995).

1977a, Ochi, 1995).

Gilbert, 1973); similar results are seen with rat kidney y-GCS (O.W. Griffith, unpublished observations).

'Rat kidney y-GCS was assayed in the presence of 10 mM~-glutamate, 10 mML-cysteine analogue,

D-Glutamate and DL-a-aminomethylsuccinate do not form peptide products (Sekura and Meister,

Values are shown for the enantiomers of P-amino-iso-butyrate with bovine y-GCS (Rathbum and

As shown in Table 3, the enzyme is inactive with L-aspartate and L-(Y-

aminoadipate, the lower and higher homologues of L-glutamate, respectively. The enzyme is active, however, with both DL-P-aminoadipate (Sekura and Meister, 1977) and DL-a-aminomethylglutarate (Sekura and Meister, 1977; Sekura et al., 1976), higher homologues that maintain glutamate-like spacing between the y-carboxylate and the amino group or the y- carboxylate and a-carboxylate, respectively. These and other data suggest that L-glutamate binds in an extended conformation and that the a-carbon region of its binding site can accomodate the folding necessary to allow p-

238 OWEN W. GRlFFITH AND R. TIMOTHY MULCAHY

aminoadipate and a-aminomethylglutarate to bind in a catalytically active conformation. Notably, both enantiomers of DL-a-aminomethylglutarate are active substrates, emphasizing the flexibility provided by insertion of - CH2-between the amino group and a-carbon (Sekura et al., 1976). P-Gluta- mate (i.e., P-aminoglutarate) is also an active substrate, again indicating that the precise spacing between the y-carboxylate and amino function is not important in 5-carbon dicarboxylic acids (Orlowski and Meister, 197 la; Sekura and Meister, 1977; Griffith and Meister, 1977). Preliminary studies suggest that P-glutamate, which is prochiral, binds in a single orientation and that only one isomer of p-glutamyl-L-a-aminobutyrate is formed (Bridges et al., 1980). The absence of either the amino group (i.e., glutarate) or the a-carboxylate (ie., y-aminobutyrate) leads to complete loss of activity as a substrate (Sekura and Meister, 1977). D-Glutamate binds and is activated by ATP, but it does not form dipeptide (Section I11 F2).

Methyl substitution of L-glutamate at its a- or P-carbon or on its amino group reduces, but does not eliminate, dipeptide formation; the P-hydroxy, but not the N-hydroxy, derivative is also active (Sekura and Meister, 1977; Griffith and Meister, 1977; Cooper and Griffith, 1979) (Table 3). These results support the view that spacial constraints near the region occupied by the amino group and the a- and P-carbons of glutamate are not too strict; N- hydroxy-L-glutamate may be inactive for nonsteric reasons (e.g., the low pK, value of its a-NHOH group). The y-methyl and y-hydroxy derivatives of glutamate bind well and are moderately strong inhibitors (see Section IIIG), but only y-hydroxyglutamate is a substrate (albeit a poor one) (Sekura and Meister, 1977). Although y-hydroxyl and y-methyl substituents are accommodated by the active site, they apparently distort the position of the reactive y-carboxylate such that dipeptide formation is either slow or impos- sible.

The rat and bovine y-GCS L-cysteine binding sites accommodate a variety of L-a-amino acids bearing relatively small hydrophobic side chains (Orlowski and Meister, 1971; Rathbun, 1967b) (Table 3). In fact, most in vitro studies with y-GCS from any source are carried out using ~ - a - aminobutyrate rather than L-cysteine as the second amino acid substrate. In all cases examined, L-a-aminobutyrate reacts with nearly the same v,, as L-cysteine, but its use avoids the cysteine-specific problems of autooxidation and thiol-mediated enzyme inactivation. Note, however, that the K, value for L-a-aminobutyrate (1.0, 2.3, and 1.3 mM for rat, human, and E. coli y-GCS, respectively) is substantially higher than the K, for L-cysteine (0.2,0.1, and approximately 0.1 mM for rat, human, and E. coli y-GCS, re-


spectively (Huang et al., 1988; Seelig and Meister, 1985a; Misra and Grif- fith, 1998), in this sense, L-a-aminobutyrate is not as "good" a substrate as L-cysteine (Ascaris suum y-GCS is exceptional, having a lower K, value for L-a-aminobutyrate (0.31 mM) than L-cysteine (0.41 mM) (Hussein and Walter, 1995). Other L-a-amino acids showing good activity as cysteine surrogates are S-methylcysteine, allylglycine, P-chloroalanine (Orlow ski and Meister, 1971b; Rathbun and Gilbert, 1973), and P-cyanoalanine (O.W. Griffith, unpublished observations). Aliphatic amino acids shorter than ~ - a - aminobutyrate (i.e., glycine and L-alanine) are poor substrates, but the im- mediate higher homologue, L-norvaline, exhibits good reactivity. Larger amino acids such as norleucine display poor activity (Orlowski and Meister, 1971b; Rathbun and Gilbert, 1973). L-Threonine, which is the P-hydroxy derivative of L-a-aminobutyrate, shows good activity as a substrate (Or- lowski and Meister, 1971b), Rathbun reports that for bovine lens y-GCS, allo-L-threonine is a five-fold better substrate that L-threonine (26% relative activity vs. 5.4%) (Rathbun and Gilbert, 1973).

The carboxylate of L-cysteine and its analogues is important for activity; neither cysteamine nor propylamine, corresponding to decarboxylated cysteine and a-aminobutyrate, respectively, is a substrate (Rathbun and Gilbert, 1973; O.W. Griffith, unpublished observations). Although bovine lens y-GCS is reportedly active with L-cysteine esters, the apparent activity is probably caused by contaminating L-cysteine formed by hydrolysis; the more stable L-cysteine amide is not a substrate (Rathbun and Gilbert, 1973). The addition of hydroxylamine to y-GCS reaction mixtures in place of L-

cysteine results in a slow formation of y-glutamylhydroxamate, probably by scavenging of the enzyme-bound intermediate, y-glutamylphosphate (Or- lowski and Meister, 1971a). Perhaps by a similar mechanism, rat y-GCS is reported to catalyze formation of y-glutamyl-Tris, i.e., y-glutamyl-tris(hy- droxymethyl)methylamide, in Tris buffers (Orlowski and Meister, 197 l b ).

D-&-Amino acids neither react nor inhibit when used in place of L-cysteine (Orlowski and Meister, 1971b; Rathbun and Gilbert, 1973), but both enantiomers of P-amino-iso-butyrate (i.e., a-methyl-P-alanine) are rnoder- ately good substrates; (Rathbun, 1967; Rathbun and Gilbert, 1973; Richman. 1975; Griffith, 1986). With the bovine enzyme, S-P-amino-iso-butyrate, corresponding to D-alanine with -CH2- inserted between the amino group and (Y-

carbon, is nearly twice as active as the R-enantiomer; the K , value of the S- isomer (13.3 mM) is, however, somewhat higher than that of the R-isomer (7.3 mM) (Rathbun and Gilbert, 1973). Other p-amino acids, including P- alanine, RS-P-amino-n-butyrate, and RS-a-ethyl-P-alanine, are neither

240 OWEN W. GRIFFITH AND R. TIMOTHY MUJXAHY

substrates nor inhibitors (Rathbun and Gilbert, 1973; Griffith, 1986). Note that R-a-ethyl-P-alanine is an analogue of L-a-aminobutyrate in the sense that R-P-amino-iso-butyrate is an analogue of L-alanine, but it is not a substrate of rat y-GCS (Griffith, 1986).

The binding of both L-glutamate analogues and L-cysteine analogues to rat y-GCS is influenced by the divalent metal ion present in the assay mixture; the results compiled in Table 3 were obtained using Mg2+. If Mn2+ is used in place of Mg2+, overall activity drops by about 75% (Onlowslu and Meister, 1971b), and the substrate specificity with respect to both glutamate analogues (O.W. Griffith, unpublished observations) and cysteine analogues (Orlowski and Meister, 1971b) becomes broader. The changes in specificity are quantitative rather than qualitative; poor substrates become better, but nonsubstrates generally remain nonsubstrates. Replacement of glutamate with its analogues can also affect the specificity of the L-cysteine site (Sekura and Meister, 1977; Sekura et al., 1976). For example, when DL- a-aminomethylglutarate is used in place of L-glutamate, the K , value for S- methyl-L-cysteine decreases from 3.1 mM to 0.53 mM (Sekura and Meister, 1977).

The specificity of rat y-GCS with respect to its nucleotide substrate has apparently not been reported. Richman (1975) found that AMP is a stronger inhibitor than ADP and that GDP, IMP, and UMP are very weak inhibitors. On the basis of this limited information, it can be predicted that specificity for ATP will be high.

2. REACTIONS CATALYZED

In addition to the ATP-dependent ligation of L-glutamate and L-cysteine to form L-y-glutamyl-L-cysteine, ADP, and Pi (reaction l), y-GCS catalyzes a variety of partial reactions, alternative reactions, and exchange reactions that provide important mechanistic insights. Orlowski and Meister (1971b) reported that the rat kidney enzyme catalyzed the hydrolysis of ATP (reaction 2) at a rate 4 1 0 % that of the overall reaction; such hydrolysis was inhibited by L-glutamate and accelerated by L-a-aminobutyrate. Subsequent studies by Sekura and Meister (1977), using enzyme purified by ATP affinity chromatography, showed that the true ATPase rate is considerably lower (about 0.5% of the overall reaction rate), but inhibition by L-glutamate and acceleration by L-a-arninobutyrate were confirmed. Inhibition of ATPase activity by L-glutamate can be understood in terms of L-y-glutamylphosphate formation (Section I11 F4). Acceleration of the ATPase reaction by L- a-aminobutyrate is modest (about 27%) but provides evidence that sub-


strates can bind to the L-cysteine site in the absence of L-glutamate, affect- ing catalytic steps in which they are not directly involved.

Reaction 1 : L-glutamate + L-cysteine + ATP yGCS - L-y-glutamyl-L-cysteine + ADP + Pi (1)

(2) Reaction 2: ATP ---+- ADP + Pi

Reaction 3: ATP + L- or D-glutamate --+- 5-oxoproline + ADP + Pi (3)

As shown in reaction 3, y-GCS also catalyzes the ATP-dependent formation of 5-oxoproline (i.e., 2-pyrrolidone-5-carboxylate, pyroglutamate) (Orlowski and Meister, 1971b). Because activation of the y-carboxylate of glutamate facilitates 5-oxoproline formation (e.g., both glutamine and glutamate y-ethyl ester spontaneously form 5-oxoproline at neutral pH) (Greenstein and Winitz, 1961), y-GCS-mediated catalysis of reaction 3 is consistent with formation of y-glutamylphosphate as an enzyme-bound intermediate (se2 Section I11 F4). Although both L- and D-glutamate yield 5- oxoproline, the reaction with D-glutamate is fourfold faster (L- and D-gluts- mate cyclize at 0.085% and 0.34% the rate of the overall reaction) (Sekura and Meister, 1977). Presumably enzyme-bound L-y-glutamylphosphate is stabilized by the active site awaiting reaction with L-cysteine, whereas D-y- glutamylphosphate fits the glutamate binding site less well, is consequently less stabilized, and cyclizes to 5-oxo-~-proline either in the active site or after dissociation. The enzyme is not able to form D-y-glutamyl-L-cysteine (Sekura and Meister, 1977).

Consistent with the reversibility of the overall reaction, y-GCS catalyzes several exchange reactions. Thus, radiolabeled L-a-aminobutyrate and L-

glutamate are incorporated into L-y-glutamyl-L-a-aminobutyrate in the presence of ADP and Pi (Orlowski and Meister, 1971b; Schandle and Rudolph, 1981); the rate of incorporation of L-a-aminobutyrate is twice that of L-glutamate (Schandle and Rudolph, 198 1). Similarly, radiolabeled ADP and Pi are incorporated into ATP in the presence of the amino acid substrates (Orlowski and Meister, 1971b; Schandle and Rudolph, 1981).

3. Steady-State Kinetics

Yip and Rudolph (1976) have carried out a detailed kinetic analysis of rat kidney y-GCS. Initial rate and inhibition studies indicate a partially random


mechanism in which Mg2+ATP binds first and L-glutamate and ~ - a - aminobutyrate (used as a L-cysteine surrogate) bind in random order to the y-GCS.Mg2+ ATP complex (Fig. 8). Consistent with the proposed mechanism, Mg2+ADP is a competitive inhibitor (Ki = 1.4 mM) with respect to Mg2+ ATP and is a noncompetitive inhibitor with respect to the amino acid substrates. L-Glutamate antagonists (e.g., L-methionine-SR-sulfoximine and a-methyl-DL-glutamate) bind competitively with respect to glutamate, noncompetitively with respect to L-a-aminobutyrate, and uncompetitively with respect to Mg*+ATP. The conclusions relating to order of substrate binding were confirmed by subsequent isotope exchange at equilibrium studies in which incorporation of L-['~C] glutamate or ~-cr-['~C] aminobutyrate into L-

y-glutamyl-L-a-aminobutyrate, or incorporation of 32Pi or [14C]ADP into ATP were quantitated (Schandle and Rudolph, 1981). Consistent with the quaternary complex mechanism shown in Figure 8, the isotope exchange studies did not indicate preferential binding of either L-glutamate or L-a- aminobutyrate to the y-GCS,Mg2+ATP complex. The authors point out, however, that small differences would be masked by the slowness of the rate-limiting interconversion of the quarternary complexes.

The order of substrate binding has also been explored by Lebo and Kredich, who found that cystamine reacted covalently to inactivate rat y- GCS (see Section IIIG) and that substrate protection from such inhibition

B C

A

E J. E + products

Figure 8. Kinetic mechanism of rat y-GCS. Diagrammatic representation of the deduced order of substrate addition to y-GCS (a. Enzyme substrate complexes are shown as EA, EAB, EAC, and EAEK, where A = ATP, B = L-glutamate, and C = L-cysteine or L-u-aminobutyrate. The order of product dissociation is not shown. Adapted from Yip and Rudolph (1976).


could be used to probe substrate binding (Lebo and Kredich, 1978). Their studies indicate that the following complexes are readily formed: y- GCS.Mg2+, y-GCS.Mg2+.MgATP2-, y-GCS.Mg2+.L-glutamate, y- GCS.Mg2+.MgATPZ-*L-glutamate, and y-GCS*Mg2+.L-y-glutamyl-L-a- aminobutyrate. The complexes of y-GCS with glutamate and y-glutamyl- a-aminobutyrate in the absence of nucleotide were not seen in the earlier steady-state kinetic studies. However, all the results are consistent with a model in which ATP binds first when ATP and glutamate are both present but, in the absence of ATP, glutamate can nonetheless bind (such binding is presumably too slow to affect the steady-state kinetics). Glutamate binding in the absence of ATP is further supported by kinetic studies with the bovine lens y-GCS (Van Buskirk et al., 1978) and by many studies showing that glutamate and Mg*+.glutamate protect y-GCS from a variety of active site directed inhibitors (see Section 111 G). As noted, the observation that L-a- aminobutyrate accelerates the ATPase reaction indicates that substrates can interact with the L-cysteine binding site in the absence of glutamate (Sekura and Meister, 1977; Orlowski and Meister, 1971).

4. Chemical Mechanism

Strumeyer and Bloch (1960) reported that y-GCS-catalyzed synthesis of y-glutamylcysteine from [UL-'80]glutamate resulted in the transfer of 1 equivalent of "0 to Pi. On the basis of that result, they proposed that the enzymatic reaction involved y-glutamylphosphate as an intermediate. Subse- quent studies confirm that view; the y-GCS reaction proceeds in two distinct chemical steps with L-y-glutamylphosphate as a tightly bound intermediate (Orlowski and Meister, 1971a,b). In the first step, the y-carboxylate of L-

glutamate attacks the y-phosphoryl of ATP to form L-y-glutamylphosphate and ADP (reaction 1A). In the second step, the amino group of L-cysteine (or L-a-aminobutyrate) attacks the y-carbonyl of L-y-glutamylphosphate to form L-y-glutamyl-L-cysteine and Pi (reaction 1B). Neither of these reactions occurs at a significant rate unless all substrates are bound, and none of the products normally dissociates until both catalytic steps are complete. However, as noted above, in the absence of L-cysteine or its surrogates, y- GCS will carry out reaction 1A with subsequent cyclization of L-y-glutamylphosphate to 5-oxo-~-proline (reaction 3). This extremely slow reaction and the more rapid formation of y-glutamylhydroxamate in the presence of hydroxylamine are easily rationalized on the basis of y-glutamylphosphate formation. Enzyme-mediated ATP-dependent formation of

244 OWEN W. GRIF’FITH AND R. TIMOTHY MULCAHY

buthionine sulfoximine phosphate is also consistent with the normal inter- mediacy of y-glutamylphosphate in the y-GCS reaction (see Section IIIG). Although y-glutamylphosphate is too unstable to allow its chemical synthesis and direct testing as an intermediate, the available indirect evidence makes a strong case for the two step y-GCS mechanism shown in reactions 1A and 1B.

Reaction ZA: L-Glutamate + ATP - L-y-glutamylphosphate + ADP

Reaction 2B: L- y-Glutamylphosphate + L-cysteine - L-y-glutamyl-L-cysteine + Pi

The presence of an unique reactive cysteine residue in the active site of most eukaryotic y-GCS has suggested to some that it may be mechanistically important. In particular, it is argued that participation of the cysteine thiol as a catalytic nucleophile would allow reaction 1B to proceed in two steps with an acyl-enzyme intermediate (i.e., HS-enzyme + y-glutamylphosphate + y-glutamyl-S-enzyme + Pi, and y-glutamyl-S-enzyme + L-cysteine + L-y-glutamyl-L-cysteine + HS-enzyme). There is, however, only circumstantial evidence in favor of this mechanism and stronger evidence against it. In support, the reactive thiol does appear to be properly located for the catalytic role proposed. Amino acid sequence alignments (Section IIIB), glutamate protection data, and reaction with y-methylene-D- glutamate (Section IIIG) all suggest that the reactive cysteine residue is conserved and is located near the y-carboxylate of bound glutamate and the y- phosphoryl of bound ATP. Proximity is thus not in question, but proximity does not establish a catalytic role. Similarly, the fact that y-GCS is inactivated by reagents blocking the cysteine residue (e.g., by reaction with cystamine) (Section IIIG) suggests that it is in the active site but does not elucidate its normal catalytic function. Steady-state kinetics are also silent on the existence or absence of catalytic steps not involving association of substrates or dissociation of products (Schandle and Rudolph, 1981; Yip and Rudolph, 1976). Against the proposed catalytic role are several observations. E. coli y-GCS catalyzes the same reaction as mammalian y-GCS with similar specific activity and substrate K , values, but it does not contain an active site thiol as judged by cystamine inhibition studies (Huang et al., 1988) and amino acid sequence alignments (Fig. 2). It seems unlikely that E. coli y-GCS would be so similar kinetically to mammalian y-GCS yet would catalyze its reaction by a chemically distinct mechanism. The fact that there is no conserved cysteine in the Leishmania y-GCS sequence (Schafer et al.,


l997), which otherwise bears moderate homology to the other eukaryotic y- GCS, also argues against the mechanistic essentiality of the active site cysteine. Finally, mammalian y-GCS are potently inhibited by S-alkyl homocysteine sulfoximines which appear to bind initially as analogues of the transition state formed when cysteine reacts with y-glutamylphosphate (see Section IIIG); the strength of their initial binding is hard to rationalize if y- glutamylphosphate reacts instead with an enzymatic cysteine residue.

To date, the only other active site residue specifically identified in mammalian y-GCS is Lys-38 of the rat heavy subunit. Thus, Chang (1996) reports that trinitrobenzene sulfonate (TNBS) inactivates rat kidney y-GCS and that the rate of inactivation is increased by Mg2+ and related metal ions. Although several amino groups are presumably modified by TNBS, Lys-38 is the only amino group modified more rapidly in the presence of Mg2+. Site-directed mutagenesis studies show that alteration of Lys-38 to Arg has little effect on activity, but replacement with Asn or Glu increases KG,'" by two- to threefold and decreases specific activity by approximately 50%. From these modest changes, Chang (1996) concludes that Lys-38 is in the glutamate binding site, perhaps contributing to binding of the carboxylate anion. Also investigating the role of basic residues, Tnoue et a]. (1993) found that of seven histidine residues in E. coli y-GCS only His-150 was essential for activity; the H150A mutant was devoid of activity. Studies defining the role of His-150 have not yet been reported.

G. INHIBITION

1. Feedback Inhibition by GSH

Following an earlier suggestion by Jackson (1969) that GSH exerted a regulatory effect on its own synthesis, Richman and Meister (1975) showed in 1974 that GSH inhibited rat y-GCS competitively with L-glutamate. It was proposed that the y-glutamyl moiety of GSH occupied the glutamate binding site preventing substrate binding4 The Ki value for GSH, reported as 2.3 mM, was comparable to tissue GSH levels, suggesting that feedback inhibition would occur. More recent studies by Huang et al. (1993b) reported a considerably higher eSH for the rat holoenzyme (8.2 mM, Table

Because GSH-mediated inhibition is competitive with L-glutamate, high intracellular concentrations of L-glutamate overcome feedback inhibition by GSH. Maede et al. report that GSH levels are abnormally h g h in canine erythrocytes that accumulate high levels of glutamate (Maede et al., 1982). By contrast, high levels of circulating L-glutamate depress GSH levels by interfering with L-cystine transport (Eck et al., 1989).

246 OWEN W. GRIFRTH AND R. TIMOTHY MULCAHY

1) but indicated that the Ki value is lower for y-GCSh (eSH = 1.8 mM). As noted earlier, they argued the higher KFSH of the holoenzyme would allow appropriate regulation of GSH synthesis, whereas y-GC&, would be relatively inactive due, in part, to strong inhibition by physiological concentrations of GSH (Huang et al., 1993b). However, the only tissue in rats with a GSH concentration approaching 8 mM is liver, with a GSH of approximately 4.5 mM); in most tissues, GSH levels are substantially lower (1-3 mM) (Grif- fith and Meister, 1979). It is thus not clear how GSH could be an effective feedback inhibitor unless glutamate concentrations were well below K, levels (i.e., < 1.4 mll/f). By contrast, for human y-GCS eSH is 3.3 mM, a value consistent with feedback regulation at physiological GSH levels (Misra and Griffith, 1998). The basis for the unusually high eSH seen in recent (Huang et al., 1993b) but not earlier (Richmond and Keister, 1975) studies with rat y-GCS requires further investigation. In our hands, eSH for rat kidney y- GCS is about 3 mM (I. Misra and 0. W. Griffith, unpublished observations).

Studies with GSH analogues indicate that the cysteine moiety of GSH is necessary for maximal y-GCS inhibition. The thiol group apparently contributes to the interaction of GSH with the enzyme at a second site distinct from the glutamate binding site. Thus, ophthalmic acid (L-y-glutamyl-L-a- aminobutyrylglycine), which is isosteric with GSH, is a relatively ineffec- tive y-GCS inhibitor when added alone (< 10% inhibition at 10 mM) but is nearly as effective as GSH in the presence of dithiothreitol (Krphthalmic acid = 11.4 mM, comparable to eSH = 8.2 mM); Huang et al. (1993b) suggest that GSH and dithiothreitol reduce the disulfide bond linking the heavy and light subunits and that this contributes to the inhibition seen. Consistent with this view, disulfide bond reduction is not an issue with the isolated heavy subunit, and dithiothreitol reportedly does not affect inhibition by either GSH or ophthalmic acid (Huang et al., 1993b). Surprisingly, however, the Ki values for GSH and ophthalmic acid as y-GCSh inhibitors are quite different (1.8 mM and 12.5 mM, respectively), and GSH inhibits competitively whereas ophthalmic acid inhibits noncompetitively (Huang et al., 1993b). It is unclear why GSH and ophthalmic acid + dithiothreitol inhibit the holoenzyme with similar affinities but interact with the isolated heavy subunit quite differently.

2. S-Alkyl Homocysteine Sulfoximine Inhibitors

Glutamine synthetase catalyzes the ATP-dependent ligation of L-glutamate and ammonia to form L-glutamine, ADP, and Pi. The reaction is mech-


anistically similar to that catalyzed by y-GCS, and, like y-GCS, involves y- glutamylphosphate as an enzyme-bound intermediate (Tate and Meister, 1973). During the early 1970s, Meister and colleagues showed that glutamine synthetase was strongly inhibited by methionine sulfoximine (MSO). Initial binding of MSO was competitive with L-glutamate and reversible, but in the presence of Mg2+ ATP, glutamine synthetase catalyzed phosphorylation of the sulfoximine N to form methionine sulfoximine phosphate, a species bound so tightly that it cannot be dissociated without dena- turing the enzyme (Tate and Meister, 1973). Richman et al. (1973) extended this work to rat kidney y-GCS, showing that it too was inhibited by MSO in the presence of Mg2+ ATP (Richman et al., 1973). As with glutamine synthetase, glutamate reduced the rate of inhibition, suggesting that MSO bound initially as a glutamate analogue. Palekar et al. (1975) showed that MSO could inhibit y-GCS (as well as glutamine synthetase) in vivo and that such inhibition led to depletion of tissue GSH and glutamine stores (Tate and Meister, 1973). Because mammalian GSH metabolism was poorly understood at the time, identification of an agent capable of pharmacologically manipulating yGCS activity and tissue GSH levels was of considerable importance. Unfortunately, when given at the doses needed to inhibit y-GCS, MSO is highly toxic, causing severe audiogenic seizures and death within several hours. These effects were attributed to derrangements of brain glutamine metabolism, but toxicity caused by inhibition of brain y-GCS could not be excluded.

Griffith et al. (1978) noted that the glutamate analogue specificity of glutamine synthetase (Tate and Meister, 1973) and y-GCS (Sekura and Meis- ter, 1977; Orlowslu and Meister 1971b) differed significantly and, on the basis of those differences, designed and synthesized a variety of MSO analogues intended to selectively inhibit either y-GCS or glutamine synthetase (Griffith and Meister, 1978; Griffith et al., 1979). Although the differences in glutamate analogue specificity proved difficult to exploit, the synthetic effort ultimately identified a-ethyl-DL-methionine-SR-sulfoximine (a- ethyl-MSO) as a selective inhibitor of glutamine synthetase (Griffith and Meister, 1978) and S-propyl-DL-homocysteine-SR-sulfoximine (DL-prothionine-SR-sulfoximine, DL-SR-PSO) as a selective inhibitor of y-GCS (Grif- fith et al., 1979). When administered to mice, DL-SR-PSO caused depletion of tissue GSH, but not of glutamine, and there was no toxicity (Griffith et al., 1979). By contrast, tissue GSH levels were essentially unchanged in mice given a-ethyl-MSO, but tissue glutamine levels decreased markedly. Mice given a-ethyl-MSO suffered convulsions and died (Griffith and Meis-

248 OWEN W. GRFF’ITH AND R. TIMOTHY MULCAHY

ter, 1978). Subsequent work established that S-butyl-DL-homocysteine-SR- sulfoximine (m-buthionine-SR-sulfoximine, DL-SR-BSO) was at least as selective for y-GCS as was DL-SR-PSO and was about 100-fold more potent (Griffith and Meister, 1979). The next higher homologue, pentathionine sulfoximine, displays an activity comparable to that of BSO but offers no advantage over BSO (Griffith, 1982). Its use is discouraged by the observation that the next higher homologues (i.e., the S-hexyl and S-heptyl derivatives) exhibit potency and selectivity for y-GCS comparable to those of BSO but are toxic for reasons not yet elucidated (Griffith, 1982). It is reported that S- isopentyl homocysteine sulfoximine has improved selectivity for y-GCS over glutamine synthetase compared with BSO (Anderson and Meister 1995), but it has not yet been shown that the improvement is of practical advantage.

Because the a-carbon and S of BSO are chiral and unresolved, DL-SR- BSO is a mixture of four isomers. Previous work with MSO (Tate and Meis- ter, 1973) suggested, however, that only the L-S-isomer of BSO was likely to be a strong inhibitor. Griffith (1982) reported an improved synthetic route, allowing a 2-isomer mixture, L-buthionine-SR-sulfoximine (L-SR- BSO), to be made in three steps from L-methionine. This material is now commercially available, as is also DL-SR-BSO. Campbell et al. (1991) reported the analytical and preparative resolution of L-SR-BSO to give gram quantities of L-buthionine-S-sulfoximine (L-S-BSO, (Fig. 9) and L-buthionine-R-sulfoximine (L-R-BSO) (Campbell et al., 1991). Only L-S-BSO was found to be a potent inhibitor of mammalian y-GCS; its ability to inhibit y- GCS is approximately twice that of L-SR-BSO and four-fold that of DL-SR- BSO. Because L-S-BSO is not yet commercially available, most reported work has been and continues to be carried out with DL-SR-BSO or L-SR- BSO. Of these, the latter is clearly refer able.^

The structural basis for the selectivity with which L-S-BSO and a-ethyl- MSO inhibit y-GCS and glutamine synthetase, respectively, has received considerable attention (Griffith et al., 1979; Griffith, 1982, 1989). As shown

The finding that BSO is active against malignant melanoma (Prezioso et al., 1992) and is useful in overcoming GSH-related resistance to therapy in other cancers (Griffith and Fried- man, 1991; Colvin et al., 1993; O’Dwyer et al., 1995; O’Brien and Tew, 1996; Schroder et al., 1996) has elicited interest in commercializing L-S-BSO, at least for clinical trials. It is possible that this material will be available also for research use. In any case, publications should spec- ify the isomeric composition of the “BSO’ used, since it affects interpretation of the results and reproducibility of the work.


L-Glutamate

FH2-CH3 +NH3-F-H

c=o 61

D3-Amiio- l-chloro-2-~tanone

LButhionine Sulfone

Figure 9. hibitors thought to bind to the L-glutamate site of y-GCS is shown.

Structures of y-GCS inhibitors. The structure of r-glutamate and of various in-

in Figure 10 bottom left), L-S-BSO binds to y-GCS as a glutamate analogue; the S-butyl group presumably extends into the L-cysteine binding site, closely mimicking the amino group and a- , p-, and y-carbons of a- aminobutyrate. By contrast, glutamine synthetase requires an acceptor substrate bindmg site only large enough for ammonia; because it cannot accomodate the S-butyl group of BSO, it is not inhibited (Fig. 10, right top and bottom). Selectivity for glutamine synthetase over y-GCS is based on the steric constraints in the a-carbon region of the glutamate binding site. Al- though both y-GCS and glutamine synthetase use a-methylglutamate as a substrate (Sekura and Meister, 1977; Orlowski and Meister, 1971b; Tate and Meister, 1973) and are inhibited by S-alkyl a-methylhomocysteine sul-

250 OWEN W. GRIFF’ITH AND R. TIMOTHY MULCAHY

7 - GLUTAMYLCYSTEINE SYNTHETASE

SUBSTRATE BOUNO

y -GLUTAMYLPHOSPHAlE 0-AMINOBUTYRATE

GLUTAMINE SYNTHETASE

O,\ c-o- I

NH3 -C-t i

y -GLUTAMYLPHOSPHATE AMMONIA

Figure 10. Binding of substrates and S-alkyl homocysteine sulfoximine inhibitors to yGCS and glutamine synthetase. (Top) Binding and reaction of y-glutamylphosphate with L - a -

aminobutyrate, a L-cysteine surrogate (lef, -y-GCS) or ammonia (right, glutamine synthetase). (Bottom) Binding of L-buthionine-S-sulfoximine phosphate to -y-GCS (leff) and the binding of a-ethyl-L-methionine-S-sulfoximine phosphate to glutamine synthetase (right). Adapted from Griffith and Friedman, 1991).

foximines (Griffith and Meister, 1978, 1979; Griffith et al., 1979; Griffith, 1982), an a-ethyl substituent can apparently be accommodated only by glutamine synthetase; thus, a-ethyl-MSO inhibits glutamine synthetase but not y-GCS (Fig. 10, bottom right).

As noted, glutamine synthetase phosphorylates the sulfoximine N of MSO to yield methionine sulfoximine phosphate, which is noncovalently but nonetheless irreversibly bound; a-ethyl-MSO also inhibits irreversibly and is presumed to react similarly (Fig. 10, lower right). Inhibition of y- GCS by BSO is mechanistically similar, in that the sulfoximine N is phosphorylated by ATP to form buthionine sulfoximine phosphate in the active site (Griffith, 1982). However, in contrast to the genuinely irreversible inhibition of glutamine synthetase seen with MSO, ATP-dependent BSO-mediated inhibition of rat y-GCS is only conditionally irreversible. If the en-


zyme is maintained in the presence of Mg2+ ATP, inhibition is nearly irreversible, but if Mg2+ ATP is removed (e.g., by dialysis or gel filtration), full activity is restored. Preservation of the tight inhibitor-enzyme interaction is apparently metal ion dependent, since Mg2+ and Mn2+ alone are moderately effective in maintaining the inhibited state (Griffith, 1982; 0. W. Grif- fith, unpublished observations). This observation suggests that BSO binding, like glutamate binding, is assisted by an active site metal ion. Although it is possible that the metal associated with ATP plays this role, it appears more likely that a second metal ion is involved. Studies addressing this issue are ongoing.

To summarize the current mechanistic view of sulfoximine-mediated y- GCS inhibition, BSO and its analogs are initially bound to the glutamate and cysteine binding sites of yGCS as transition state analogues in which the tetrahedral sulfoximine S mimics the tetrahedral intermediate formed when cysteine reacts with y-glutamylphosphate in the normal catalytic reaction. The strength of this initial binding is difficult to estimate with L-S-BSO because it is very rapidly phosphorylated to form a tightly bound species (Grif- fith, 1982; Campbell et al., 1991). However, L-R-BSO and buthionine sulfone (which has two oxygens on S, Fig. 9) have similar tetrahedral geometry but are not phosphorylated. They inhibit reversibly and competitively with g glut am ate and have Ki values are 0.15 mM and 0.06 mM, respectively (Campbell et al., 1991). These values, significantly lower than the observed K , for L-glutamate (approximately 1.5 mM), suggest that a tetrahedrally substituted S provides significant binding affinity, and support the charac- terization of L-S-BSO as a transition state analogue inhibitor, albeit a relatively weak one. More definitive conclusions await evaluation of the binding contribution of the S-butyl group. The fact that L-S-BSO is phosphorylated as a pseudosubstrate to a form a tightly bound, strongly inhibiting species establishes that that diastereomer of BSO is also a mechanism-based inhibitor (i.e., k,, inhibitor, suicide substrate).

As the prototypic y-GCS inhibitor, BSO has been tested with enzyme isolated from most species; in all cases examined it inhibits (e.g., human Sri- ram and Ali-Osman, 1993; Misra and Griffith, 1998), E. coli (Watanabe et al., 1986a; Inome et al., 1993; Huang et al., 1988), higher plants (Hell and Bergman, 1990), yeast (Dennda and Kula, 1986), protozoa (Lueder and Phillips, 1996), and nematode (Hussein and Walter, 1995). For rat (Camp- bell et al., 1991), human (I. Misra and 0. W. Griffith, unpublished observations) and E. coli (Huang et al., 1993b) y-GCS it has been established that L-S-BSO is the only BSO diastereomer that is phosphorylated and that

252 OWEN W. GRlFLlTH AND R. TIMOTHY MULCAHY

therefore inhibits strongly. Because the stereochemistry at the sulfoximine S of L-S-BSO determines the relative orientation of the L-glutamate, L-cysteine and All’ binding sites (Campbell et al., 1991) (Fig. lo), the fact that a single diastereomer of BSO inhibits all three enzymes implies that the overall active site geometry has been preserved from bacterial to mammalian y- GCS.

There are, however, some differences in the interactions of BSO and its analogues with y-GCS from different species. For example, inhibition of E. coli yGCS is weaker and much slower than is seen with rat or human enzyme. Preliminary studies suggest that initial binding of BSO is not im- paired but subsequent phosphorylation of L-S-BSO is slow (C. Donnelly, M.A. Hayward, and O.W. Griffith, unpublished observations). Slowness to form the strongly inhibiting phosphorylated derivative may account for why some investigators find that BSO is not an effective GSH-depleting agent for intact E. coli (Romero and Canada, 1991), whereas other investigators report effective inhibition (Moore et al., 1989). E. coli y-GCS is also dis- tinctive in being inhibitable by a-ethyl derivatives of MSO and BSO. Thus, a-ethyl-DL-buthionine-SR-sulfoximine has about 0.5% of the activity of L-

SR-BSO with rat kidney y-GCS but is about 20% as active as L-SR-BSO with the bacterial enzyme (R. Lurvey, M.A. Hayward, and O.W. Griffith, unpublished observations). This distinction suggests that selective inhibition of bacterial GSH synthesis should be possible. Because GSH plays a protective role in bacteria that contain it (Murata and Kimura, 1982; Apon- toweil and Berends, 1975), pharmacological depletion of GSH stores may have therapeutic value. A similar selective approach may be possible with the y-GCS from T. brucei, which have a high intrinsic dependence on GSH and its derivative, trypanothione, for survival (Lueder and Phillips, 1986; Fairlamb and Cerami, 1992; Arrick et al., 1981). It has been shown, for example, that administration of L-SR-BSO to mice infected with T. brucei brucei kills the protozoa and cures or extends the survival of the mice (Arrick et al., 1981). It was also recently reported that Ascaris is exceptionally susceptible to DL-SR-BSO-mediated GSH depletion, suggesting an additional therapeutic application (Hussein and Walter, 1996).

3. Clinical Use of BSO to Modulate GSH Levels in Cancer Patients

Administration of BSO to animals in doses of about 2 mmolkg blocks GSH synthesis in virtually all tissues; of the tissues examined, only brain is relatively spared because of the slow passage of BSO through the blood-


brain barrier (Griffith and Meister, 1979; Fekete et al., 1990). In the absence of further GSH synthesis, ongoing reactions of GSH utilization, mainly transport of GSH out of the cell, cause rapid GSH depletion in tissues having modest to high rates of GSH turnover, (e.g., kidney, liver, pancreas, and plasma are >50% depleted in <2 h) (Griffith and Meister, 1979). Where GSH turnover is slow (e.g., red blood cells), short-term inhibition of GSH synthesis is not accompanied by significant GSH depletion. Because GSH was known to play a protective role in tumor cells, Meister and Griffith (1979) suggested that BSO might sensitize tumors to radiation and chemotherapy. Subsequent preclinical studies with animals bearing drug-resistant tumors convincingly demonstrated that intracellular GSH levels in tumor cells can be reduced by administration of BSO at levels that are them- selves nontoxic (Griffith and Friedman, 199 1). Although GSH depletion also occurs in normal tissues (Griffith and Meister, 1979; Lee et al., 1987), the magnitude and kinetics of GSH depletion and repletion were tissue dependent and, in many cases, recovery from GSH depletion was slower in tumors than in relevant normal tissues. The possibility that tumors could be selectively sensitized to radiation or chemotherapeutic agents was evident (Griffith and Friedman, 1991; Lee et al., 1987). Indeed, the reduction in tumor GSH levels correlated with improved tumor responsiveness to several alkylating agents and cisplatin with only minimal increase in adverse normal tissue reactions. On the basis of these results, phase I clinical trials were carried out to establish optimal scheduling for drug administration and to as- sess the toxicity and biological effectiveness of L-SR-BSO in a therapeutic setting (Bailey et al., 1994; O’Dwyer et al., 1996). After phase I toxicity testing, limited phase I1 studies have been initiated to determine the efficacy of BSO in combination with melphalan (L-PAM), a DNA-crosslinking, bifunctional alkylating agent, for ovarian cancer and malignant melanoma.

Although the results of the phase I1 trial are not yet available, the phase I trials establish that biologically effective doses of BSO can be administered to patients with minimal toxicity (Bailey et al., 1994; O’Dwyer et al., 1996). In initial studies conducted by Mulcahy and coworkers, L-SR-BSO (1 5 1 3 . 1 g/m2) was administered as a 30-min infusion every 12 h for up to 10 doses; toxicity was limited to mild nausea and vomiting (Bailey et al., 1994). Glutathione levels in peripheral blood leukocytes (PBLs) were typically reduced by 60-90%, although the extent of depletion was highly variable and was not dose dependent. Pharmacodynamic studies established that y-GCS inhibition in PBLs was maximal near the completion of each 30-min BSO infusion with almost complete recovery of y-GCS activity to pretreat-

254 OWEN W. GRIFFITH AND R TIMOTHY MULCAHY

ment levels prior to the next scheduled dose; this pattern mirrored plasma BSO levels, which declined with a terminal half-life (tIl2) of 2 h (Bailey et al., 1994). Interestingly, differential elimination of the L-R and L-S-diastere- omers of BSO was observed, with the inactive LR-isomer being cleared more rapidly (Bailey et al., 1994; Gallo et al., 1995) (C1,b: L-R-BSO = 153 ml/min/m2 vs. L-S-BSO = 102 ml/min/m2; P < 0.0001) (Bailey et al., 1994).

In treatment courses including melphalan, the alkylator was administered 1 h after the last or penultimate BSO infusion. Comparison of toxicities in paired courses in individual patients shows that BSO increased melphalan- induced myelosuppression, but the combination was otherwise well tolerated (Bailey et al., 1994, O’Dwyer et al., 1996). The increase in normal tissue toxicity was accompanied by an decrease in the rate of melphalan clearance in courses including BSO (Bailey et al., 1994), but the precise mechanism accounting for altered melphalan pharmacokinetics has not been elucidated. Although phase I trials are not designed to evaluate efficacy, the combination of BSO and melphalan produced clinical responses in a limited number of previously refractory patients, supporting the view that BSO-induced GSH modulation may represent a novel and effective therapeutic strategy (Bailey et al., 1994; O’Dwyer et al., 1996).

Analysis of the phase I pharmacodynamic data suggested that intermit- tent bolus infusions of BSO does not represent an optimal schedule; recovery of y-GCS activity during nadirs of plasma and, presumably, tumor BSO levels potentially allows restoration of GSH levels toward normal before administration of a subsequent bolus of BSO. To address this problem, Bailey and colleagues conducted a second phase I trial employing a continuous (24-72 hr) intravenous infusion of BSO. Continuous infusion of BSO (0.75 or 1.5 g/m2/h for 6 7 2 h after an initial loading dose of 3.0 g/m2) was not associated with significant toxicity, was well tolerated by patients, and maintained steady state plasma BSO concentrations of approximately 500 pM and 1000 pM when the infusion rates were 0.75 or 1.5 g/m2/h, respectively (Bailey et al., 1997). Activity of PBL -y-GCS decreased by 40-70% and remained low for the duration of the infusion, recovering to baseline levels within 24 h of termination of BSO infusion. This schedule caused dramatic reductions (>90%) in GSH levels measured in tumor biopsy samples ex- cised from patients during the course of BSO infusion. Administration of melphalan near the end of the infusion schedule was well tolerated. On the basis of these results, ongoing trials will use a regimen consisting of a 30- min loading infusion of L-SR-BSO at 3.0 g/m2 followed immediately by a


72-h continuous infusion at 0.75g/m2/h; intravenous melphalan is given 48 h after initiation of BSO infusion.

4. Inactivation by Cystamine

Mammalian y-GCS is very rapidly inhibited by cystamine (NH2-CH2- CHZ-S-S-CH2-CHz-NH2, Fig. 9) as first reported by Griffith et al. (1977) and by Lebo and Kredich (197Q6 With human erythrocyte y-GCS the rate constant for cystamine-mediated inactivation is 1020 min-' mM-' (i.e., tl/2

- 4 s under pseudo-first-order conditions with 10 pM cystamine). L-Gluta- mate and Mg2+ offer substantial protection (Lebo and Kredich, 1978, Grif- fith et al., 1977), the rate constant for inactivation is reduced to 5.2 min-' mM-' by saturating Mg2+ and to <O. 1 min-' mM-' by saturating L-glutamate + Mg2+. If cystamine is added to substrate-containing reaction mixtures (e.g., mixtures used to assay activity), inactivation proceeds at mea- surable rates with cystamine concentrations ranging from about 10 pM to l mM. Because cystamine causes inactivation rather than reversible inhibition, assay progress curves are concave downward and conventional steady- state kinetics do not apply. Determination of Ki values for cystamine, repre- senting its equilibrium binding to y-GCS before covalent reaction, is difficult and requires great care to ensure that initial rates are being measured.

Inactivation by cystamine results in the formation of a mixed disulfide between a unique enzymatic cysteine residue and cysteamine (i.e., enzyme- S-S-CH2-CH2-NH2 ). As expected, inactivation is not reversed by removal of unbound inhibitor, but is readily and completely reversed by addition of thiols such as dithiothreito.' Although definitive identification of the reactive cysteine has not been reported, sequence analyses identify a conserved cysteine at position 250 in the rat y-GCS sequence (see Section IIIB), and all tested y-GCS containing the conserved cysteine, (e.g., rat (Griffith et al., 1977), human (Lebo and Kredich, 1978), T. brucei and (Lueder and Phillips, 1996), and Arabidopsis (M. May, personal communication), are inhibitable by cystamine (the yeast and Leishmania enzymes have apparently not been

Jackson had earlier reported that human erythrocyte yGCS was inhibited by cysteamine (Jackson, 1969). In retrospect, the inhibition observed was undoubtably due to cystamine, which is rapidly formed by autooxidation of cysteamine. ' Note that prolonged exposure to dithiothreitol or other thiols causes an irreversible inac-

tivation of rat 7-GCS. Reduction of the disulfide bond linking the heavy and light subunits may contribute to this effect, but other reactions, not yet elucidated, are apparently involved as well.


tested). By contrast E. coli y-GCS lacks the conserved cysteine and is not inactivated or inhibited by cystamine in a dithiothreitol-reversible manner (high concentrations (10 mM) slowly inactivate by an unknown mechanism) (Huang et al., 1988).

It is notable that the reactive cysteine of rat y-GCS, while easily modified by cystamine, reacts slowly or not at all with other common thiol reagents. For example, 5,5'-dithio-bis(2-nitrobenzoate) modifies a single cysteine residue but does not affect activity (Seelig and Meister, 1984), and iodoacetamide inactivates the enzyme only slowly (Huang et al., 1988). p - Hydroxymercuribenzoate and p-chloromercuribenzenesulfonate are more potent inhibitors than iodoacetamide (Orlowski and Meister, 1971) but inactivate more slowly than cystamine; the residue modified is unknown.

As discussed in Section IIIF, substrate protection data indicate that cystamine reacts with an enzymatic cysteine residue located in the L-glutamate binding site, probably near the region occupied by the y-carboxylate and putative second metal ion (Lebo and Kredich, 1978). Although the structural basis by which the glutamate binding site recognizes cystamine remains to be elucidated, it is clear that structural constraints apply to only one end of the cystamine molecule (Seelig and Meister, 1982). For example, NH2- CH2-CH2-S-S-CH3 and NH2-CH2-CH2-S-S-CH2-NH-Sepharose both react covalently with rat y-GCS. With the latter compound, the active site cysteine residue can apparently react with either S of the disulfide bond, be- coming attached either to cysteamine or to the resin, cysteamine-Sepharose (Seelig and Meister, 1982). Considering these findings and the fact that L-

glutamate and Mg2+ protect (Lebo and Kredich, 1978; Griffith et al., 1977), it is suggested that cystamine binds as a partial glutamate analogue, using the a-amino group binding site and extending into the region occupied by the glutamate side chain; this model places the cystamine S atoms in the positions normally occupied by C-4 and C-5 of glutamate. Although this is currently the best available model for cystamine binding, other data are not easily accommodated, including the following: (1) cystine does not inhibit and yet it would seem capable of binding in a cystamine-like manner to y- GCS, using the glutamate a-carboxylate binding site for its own carboxylate; (2) Bunte salts of cysteine and homocysteine (e.g., HOOC-CH(NH2)- (CH2)x-S-S03-, where x = 1 or 2) inhibit, but do not react covalently with an active site cysteine, even though they are very susceptible to attack by thiols (Moore et al., 1987); and (3) derivatives of cystamine in which both amino groups are substituted by bulky groups are effective y-GCS inactiva- tors (Schor, 1988; Schor et al., 1990). It seems unlikely that similarly sub-


stituted glutamate analogues would be accommodated by the binding site, although such compounds have apparently not been tested. Additional studies are necessary to establish definitively the structural basis of cystamine recognition.

Inhibition of y-GCS by cystamine analogues became of significant clinical interest following the observation by Schor (1987, 1988a) that many radioprotective and chemoprotective agents structurally related to cystamine inhibit y-GCS both in vitro and in vivo, causing GSH depletion and sensiti- zation to toxic species. For example, WR2721 (i.e., NHz-(CH&-NH-CH~- CH2-S-P03H2) is hydrolyzed in vivo to the corresponding thiol, N-2-mercaptoethyl-l,3-diaminopropane (MDP), which is a proposed radioprotective agent. However, oxidation of MDP to the disulfide produces a cystamine analog that potently inhibits y-GCS; formation of the disulfide is facilitated by reactive oxygen species such as those generated by redox cycling anti- cancer drugs or radiation (Schor, 1988b). Schor and colleagues have elucidated some of the structural constraints on cystamine analog-mediated y- GCS inhibition in an effort to design radio- and chemoprotective agents that will not cause counterproductive GSH depletion. The addition of substitu- tents near S decreases inhibition, but the addition of even quite bulky substituents to the amino group does not reliably decrease inhibition and may even increase inhibition over that seen with cystamine (Schor et al., 1990).

5. Inactivation by Chloroketones and Related Compounds

5-Chloro-4-oxo-~-norvaline(~-2-amino-4-oxo-5-chloropentanoate; Khedouri chloroketone, Fig. 9) was originally identified as a glutamine antagonist able to inactivate glutamine-dependent carbamyl phosphate synthetase (Khedouri et al., 1966). Appreciating that it might also function as a glutamate antagonist, Sekura and Meister (1977) demonstrated that it bound to and irreversibly inactivated rat y-GCS. Binding was reduced by L-glutamate, modestly increased by L-a-aminobutyrate, and showed complete dependence on divalent metal ions (Mg2+, Mn2+, Ca2+, Cd2+, or S?'); approximately 3 p,M Mg2+ was required to achieve a half-maximal rate of inactivation. Kinetic analyses indicated that initial binding was reversible (Ki = 7.75 mM) and that inactivation proceeded with a rate constant of 10.2 min-'. Radiolabeled inhibitor bound stoichiometrically and exclusively to the heavy subunit (Sekura and Meister, 1977). Although the residue modified has not yet been identified, it has been hypothesized to be the cysteine modified by cystamine (Beamer et al., 1980).


Beamer et al., (1980) explored the possibility of inhibiting rat y-GCS with chloroketone inhibitors related to L-a-aminobutyrate. Both D- and L-3- amino- 1-chloro-2-pentanone (i.e., CH3-CH2-CH(NH2)-CO-CH2C1, Fig. 9) proved to be good inhibitors, but L-glutamate rather than L-a aminobutyrate protected against inactivation. These compounds thus resemble 5-chloro-4- 0x0-L-norvaline in interacting with the L-glutamate binding site prior to covalent reaction. Consistent with this view, prior reaction with either cystamine or BSO completely protects y-GCS from covalent modification with 3-amino-1 -chloro-2-pentanone. The a-aminobutyrate-based chloroketones are significantly more potent than 5-chloro-4-oxo-~-norvaline; 20 pM D- or L-3-amino- I-chloro-2-pentanone inhibits comparably to 1 mM 5-chloro-4- 0x0-L-norvaline. Inhibition by D-3-amino- 1 -chloro-2-pentanone was unaf- fected by divalent metal ions, but inhibition by the L-enantiomer was modestly reduced by Mn2+. Because these inhibitors bear little structural resemblance to L-glutamate, it may be more accurate to view them as cystamine analogues (i.e., the covalently reactive chloroketone carbon is two carbons removed from the amino group just as a reactive S of cystamine is two carbons removed from its amino group). If this model is correct, D- and L-3-amino- 1 -chloro-2-pentanone may also modify the uniquely reactive active site cysteine residue. Additional work is necessary to address this issue and to clarify the structural basis for the initial interaction of these chloroketones with y-GCS.

Other chloroketones have not been systematically examined for their ability to inhibit y-GCS. The simplest chloroketone, chloroacetone, inhibits, but must be added at 10 mM to achieve, levels of inhibition seen with 20 p,M 3-amino-1-chloro-2-pentanone (Beamer et al., 1980). As noted, iodoacetamide also inhibits when added in mM concentration. Inhibition by both chloroacetone and iodoacetamide is prevented by prior reaction with cystamine, again suggesting that inactivation may occur only when the active site cysteine residue is modified.

6. Other Glutamate Antagonists

Rat y-GCS is inhibited by a wide range of glutamate antagonists (Sekura and Meister, 1977; Griffith and Meister, 1997) many of which are also alternative substrates that react more slowly than does L-glutamate (Table 3). Among the more interesting of these compounds are those such as L-homocysteine sulfinate, L-homocysteine sulfonate, and ~~-2-amino-4-phospho- nobutyrate, which replace the y-carboxylate of L-glutamate with -SO, S O , and PO, respectively (Moore et al., 1987; Marche et al., 1987). Each of


these groups is tetrahedral and can thus potentially mimic the tetrahedral transition state formed when y-glutamylphosphate reacts with cysteine. Each is a moderately good inhibitor that binds competitively with L-glutamate; the sulfinate, sulfonate, and phosphonate have Ki values of 0.27 mM, 0.58 mM, and 0.20 mM, respectively (Moore et al., 1987).

As noted in the discussion of cystamine, the Bunte salts of cysteine and homocysteine (i.e., the S-sulfo derivatives) (Fig. 9) are potent inhibitors, although they do not react covalently with the active site cysteine residue (Moore et al., 1987). Both the L- and D-enantiomers of these inhibitors are active, and S-sulfocysteine is somewhat more potent than S-sulfohomocys- teine. Initial binding is competitive with glutamate but is not particularly strong (Ki values range from 2.7 to >50 mM). Initial binding is followed, however, by a slow, metal ion-dependent, nearly irreversible inactivation of the enzyme. Because L-homocysteine sulfonate, which is isosteric with S- sulfo-L-cysteine, does not cause inactivation, Moore et al. (1987) concluded that the sulfenyl S must permit a structural rearrangement in the inhibitor that results in very tight but noncovalent binding; these investigators suggest that HOOC-CH(NH2)-CH2-S-SOT, when bound, may undergo an elec- tronic rearrangement to HOOC-CH(NH2)-CH2-S+ = SO:; with the latter tightly bound.

Although yGCS binds D-glutamate and converts it to 5-oxo-~-proline, D-enantiomers of glutamate analogues are, in general, less effective inhibitors than the L-enantiomers. The exception to this rule is 4-methylene- D-glutamate (Fig. 9), which was shown by Simondsen and Meisten (1986) to inactivate rat y-GCS. Inactivation was metal ion dependent (Mn2+ > Mg2+) and was accompanied by covalent binding of radiolabeled 4-methylene-D-glutamate to the enzyme. Studies in which the inactivated enzyme was hydrolyzed and the resulting amino acids fractionated suggested that the inhibitor reacts as a Michael acceptor with an enzymatic cysteine residue. Because the reactive double bond of 4-methylene-~-glutamate would be positioned near the presumed location of the reactive active site cysteine, that residue is thought to be the one modified. 4-Methylene-~-glutamate was a weak competitive inhibitor (Ki = 2.5 mM) but did not cause inactivation.

7. Inhibition by Nitric Oxide Donors

Both protein and low-molecular-weight thiols can be nitrosylated by nitric oxide (NO) in the presence of an electron acceptor (Gow et al., 1997) or by direct nitrosylating species such as N203 and other nitrosothiols (RSNO)


(Wink et al., 1996). Such reactions have been shown to occur in intact cells and in vivo and typically result in activation or inactivation of the enzymes or receptor proteins modified. In view of the high reactivity of the active site cysteine of y-GCS, Han et al. (1995) examined its susceptibility to modification by NO donors. Rat y-GCS is inactivated by S-nitroso-L-cysteine and S-nitroso-L-cysteinylglycine, but not by S-nitroso-GSH. It is also inactivated, albeit more slowly, by NONOates, reagents that spontaneously re- lease NO. Inhibition is completely prevented by pretreatment of the enzyme with L-SR-BSO and ATP (reactivation by gel filtration in the absence of Mg2+ ATP) and is largely prevented by pretreatment with cystamine (reactivation with dithiothreitol). Mouse peritoneal macrophages treated with cy- tokines to induce expression of nitric oxide synthase show increased NO formation, inhibition of GSH synthesis and decreased GSH levels (Han et al., 1995). The sensitivity of y-GCS to inhibition by NO donors may compromise cellular defenses against oxidative and nitrosative stress at sites at which NO production is increased (e.g., at sites of inflammation).

IV. Conclusions and Perspective

The past 25 years have witnessed an enormous expansion of our knowl- edge of GSH biosynthesis in both mammalian and nonmammalian systems, but the quest is far from complete. The enzymology of the key rate-limiting enzyme, y-GCS, is now well elucidated in terms of its catalytic mechanism, kinetics, and substrate specificity, but we know little about precisely how the enzyme facilitates the formation of y-glutamylphosphate and the further reaction of that intermediate with cysteine to form GSH. It is anticipated that the recent cloning and expression of the rat, human, E. coli, and T. brucii y- GCS proteins will permit more intense probing of mechanism and structure, including X-ray crystallographic and site-directed mutagenesis studies.

Understanding the regulation of GSH synthesis is central to appreciating the role of this important peptide in health and disease. Feedback inhibition of y-GCS by GSH was reported as early as 1969, but it was only during the past 5 years that the mechanism of inhibition and the key role of the y-GCS light subunit were elucidated. Similarly, recent analyses of the y-GCS ge- netic sequences provide the first insights into how oxidative stress and other physiological and pathophysiological stresses increase y-GCS transcription, increasing the rate of GSH synthesis. Additional studies are necessary to establish the relative importance of specific transcriptional control mechanisms under various conditions. The possibility that yGCS is coordinately


expressed with other enzymes and transport systems involved in GSH synthesis and metabolism warrants particular attention.

Finally, the demonstration that elevated GSH levels can mediate resistance to radiation and chemotherapy in cancer treatment has greatly stimulated interest in the pharmacological control of GSH biosynthesis. Buthion- ine sulfoximine, the best agent available for reducing GSH levels in vivo, has entered clinical trial and been shown effective as a relatively nontoxic GSH depleting agent. Whether it will provide significant therapeutic benefit remains to be established. Independent of the outcome of those trials, it is likely that the development of more potent and perhaps tumor-specific y- GCS inhibitors and a more complete understanding of precisely how GSH contributes to resistance will ultimately prove beneficial to cancer patients. Additional applications of y-GCS inhibitors in the treatment of parasitic diseases such as trypanosomiasis have also been identified. It is likely that a better understanding of the role of GSH in other infectious diseases as well as in inflammatory and autoimmune pathologies will establish still additional applications for agents able to manipulate GSH biosynthesis or metabolism.

Acknowledgments

Studies from the authors’ laboratories were supported in part by National Institutes of Health grants DK26912 (to O.W.G.), CA77233 (to O.W.G.), and CA57549 (to R.T.M.). The authors thank the many colleagues who con- tributed to the work summarized here, in particular Ila Misra, Ph.D., Jerry J. Gipp, Ph.D., Ernest B. Campbell, and Michael A. Hayward.

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