Glutathione biosynthesis-Overview.pdf

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Chemico-Biological Interactions 111112 (1998) 114

Glutathione: an overview of biosynthesis andmodulation

Mary E. Anderson *

Department of Microbiology and Molecular Cell Sciences, The Uniersity of Memphis, Memphis,

TN38152,USA

Abstract

Glutathione (GSH; -glutamylcysteinylglycine) is ubiquitous in mammalian and other

living cells. It has several important functions, including protection against oxidative stress.

It is synthesized from its constituent amino acids by the consecutive actions of -glutamyl-

cysteine synthetase and GSH synthetase. -Glutamylcysteine synthetase activity is modulated

by its light subunit and by feedback inhibition of the end product, GSH. Treatment with an

inhibitor, buthionine sulfoximine (BSO), of -glutamylcysteine synthetase leads to decreased

cellular GSH levels, and its application can provide a useful experimental model of GSH

deficiency. Cellular levels of GSH may be increased by supplying substrates and GSH

delivery compounds. Increasing cellular GSH may be therapeutically useful. 1998 Elsevier

Science Ireland Ltd. All rights reserved.

Keywords: Glutathione; -Glutamylcysteine synthetase; GSH synthetase; 2-Oxothiazolidine4-carboxylate; GSH esters

1. Introduction

This chapter seeks to give a brief overview of glutathione function, synthesis and

modulation of its intracellular levels. There are too many papers on glutathione to

cite them all, but for reviews see [1 6]. Many of the pioneering studies on glutathione

synthesis, metabolism and function were led by the late Dr Alton Meister.

* Tel.: +1 901 6782985; fax: +1 901 6784457; e-mail: [email protected]

0009-2797/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved.

PII S0009-2797(97)00146-4


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M.E. Anderson/Chemico-Biological Interactions 111112 (1998) 1142

Fig. 1. The Stucture of glutathione (GSH); L--glutamyl-L-cysteinylglycine.

Glutathione (GSH) is a tripeptide of glutamate, cysteine and glycine that

contains an unusual -peptide bond between glutamate and cysteine (Fig. 1). Such

a bond prevents GSH from being hydrolyzed by most peptidases. GSH is less easily

oxidized than its precursors, cysteine and -glutamylcysteine. GSH is found in most

mammalian and many prokaryotic cells and is the most abundant intracellular thiol

(0.210 mM). Intracellularly, GSH is kept in its thiol form by glutathione disulfide

(GSSG) reductase, a NADPH-dependent enzyme. GSH has several important

cellular functions (Fig. 2). GSH participates as a coenzyme and is involved inamino acid transport. It is involved in metabolism and the maintenance of the thiol

moieties of proteins and low molecular weight compounds, such as cysteine and

coenzyme A. GSH is also involved in maintaining ascorbic acid in its reduced form

and in the formation of deoxyribonucleotides. GSH reacts enzymatically (GSH

S-transferase family) or non-enzymatically with toxic compounds to form GSH

Fig. 2. Overview of glutathione (GSH) metabolism.


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M.E. Anderson/Chemico-Biological Interactions 111112 (1998) 114 3

conjugates. It also protects against oxidative damage caused by reactive oxygen

species (ROS) that may be formed normally in metabolism. GSH may react with

ROS non-enzymatically. Hydrogen peroxides and other peroxides are detoxified by

GSH peroxidase.

2. GSH biosynthesis

GSH is synthesized intracellularly by the consecutive actions of -glutamylcys-

teine (reaction 1) and GSH (reaction 2) synthetases:

L-Glu+L-Cys+ATPL--GluL-Cys+ADP+Pi (1)

L--Glu-Cys+Gly+ATPGSH+ADP+Pi (2)

Cysteine is usually the limiting substrate in the synthesis of GSH. Intracellular

GSH is exported from most cells, but it is not significantly taken up by cells under

normal conditions [5]. Once outside of the cell, the -glutamyl bond of GSH may

be cleaved by the membrane bound -glutamyl transpeptidase whose active site is

on the outside of some cells/organs. Transpeptidase is found in the kidney, choroidplexus, lymphocytes, biliary duct, ciliary body, intestine and pancreas. The product

of the reaction is a -glutamyl enzyme which can accept an amino acid to form

-glutamyl amino acid. After transport, the -glutamyl amino acid is cleaved by

-glutamyl cyclotransferase to yield free amino acid and 5-oxoproline (a cyclic form

of glutamate). 5-Oxoproline is ring opened by 5-oxoprolinase to give glutamate.

The biosynthetic enzymes, together with these latter three enzymes, form the

-glutamyl cycle [7]. One of the best acceptor amino acids for transpeptidase is

cystine; thus, its product is -glutamylcystine [8]. This may be transported into

certain cells, such as kidney, and reduced to cysteine and -glutamylcysteine.

Cysteine can be used to synthesize GSH using reactions (1) and (2) or be used for

other cellular needs. -glutamylcysteine can be used directly by GSH synthetase

(reaction 2) to form GSH, bypassing the first enzyme. These series of reactionsconstitutes the alternative or salvage pathway of GSH biosynthesis [2].

2.1. -Glutamylcysteine synthetase

-Glutamylcysteine synthetase has been purified from a variety of sources

[2,9,10]. The E. coli enzyme has been cloned and sequenced and is a single

polypeptide chain [11]. The rat kidney enzyme was the first mammalian form to be

cloned and sequenced [10]; it is a heterodimer. The enzymes from both sources

catalyze the same reaction, have similar apparent Km values, turnover numbers,

substrate specificity, are feedback inhibited by about the same amount, but there is

no significant sequence homology between the E. coliand the heavy subunit of the

kidney enzyme [10]. The rat kidney and human [12] enzyme, however, are highly

homologous.


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The E. coliand rat enzymes are feedback inhibited by GSH [13,14], but not by

its non-thiol analog ophthalmic acid (-glutamyl--aminobutyrylglycine). The ap-

parent Ki value of GSH for the rat kidney enzyme is about 1.5 mM [13,14]. This

inhibition is non-allosteric and competitive with glutamate. Intracellular GSH levels

are high, for example, about 4 mM for mouse kidney, while those for glutamate are

about 3 mM. These findings suggest that -glutamylcysteine synthetase is probably

not acting at its maximal rate in vivo. Both the rat kidney and the E. colienzyme

are also inhibited by amino acid sulfoximines that are transition state analogs, suchas L-prothionine-SR-sulfoximine [15] and L-buthionine-SR-sulfoximine (BSO) [16].

The rat kidney, but not the E. coli enzyme, is also inhibited by cystamine [17],

D--methylene glutamate [18], S-sulfocysteine and S-sulfohomocysteine [19], L-

amino-4-oxo-5-chloropenoate [20] and D- and L-3-amino-1-chloro-2-pentanone [21].

These studies suggested that the enzyme has an active site thiol at or near its active

site.

The heavy subunit of the rat kidney enzyme is catalytically active [22,23];

however, the apparent Km value for glutamate is much higher (13 times) than

that of the holoenzyme [23]. The light subunit has no catalytic activity, but when

co-expressed or separately expressed and mixed with the heavy subunit, the

apparent Kmvalue for glutamate returns to about the normal value [24]. While both

the heavy subunit and the holoenzyme are inhibited by GSH, the heavy subunit ismore sensitive to GSH inhibition [23]. In contrast to the holoenzyme, the heavy

subunit is inhibited by ophthalmic acid. When the holoenzyme was pretreated with

dithiothreitol, it was inhibited by ophthalmic acid [23], suggesting that a reductive

step is necessary for inhibition and that the light subunit has a regulatory function.

The two subunits cannot readily be separated by native gel filtration and only

partially separated by native gel electrophoresis under highly reducing conditions

[2224]. After isolation, about 70% of the enzyme is found to be disulfide linked

[23]. Mild treatment with GSH (1 min., 1.5 mM) followed by SDS-PAGE, in the

absence of dithiothreitol, leads to about a 50% dissociability of the subunits. In

contrast, harsher treatment with dithiothreitol (10 mM; 60 min.) or 2-mercap-

toethanol (0.7M, 5 min, 100C) are required to obtain the same dissociability as

with 1.5 mM GSH for 1 min. The interaction of GSH with the enzyme is not

apparently via disulfide bond formation because the interactions are reversible by

dialysis and even when radioactive GSH is used, no labeled enzyme could be

obtained [23]. These findings suggest that the two subunits are tightly bound by

hydrophobic interactions.

2.2. GSH synthetase

GSH synthetase catalyzes the ATP-dependent formation of GSH from -glu-

tamylcysteine and glycine. It has been purified from a variety of sources [2,9,25

27]. TheE.colienzyme is a tetramer of identical subunits (Mr, 35,559) and has been

extensively studied [28 34]. The rat kidney enzyme was the first mammalian

glutathione synthetase to be cloned and sequenced [35]. It is composed of two

identical subunits (Mr, 52,344) and contains about 2% carbohydrate (neutral sugar,


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amino sugar and sialic acid) [26]. There are two asparagine residues and one serine

residue that match the signature for glycosylation. Like -glutamylcysteine syn-

thetase, rat kidney GSH synthetase shares no significant homology between the E.

colienzyme. There is similarity with the fission yeast (about 30%) [36], putative frog

(65%) [37] and human (88%) [38]. The rat kidney enzyme is inhibited by p-

chloromercuribenzoate like the E. coli enzyme, but it is not inhibited by either

N-ethylmaleamide or 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) as is the E. coli

enzyme. Recent studies [39] with the human enzyme fusion protein suggest thatCys-422 is important for enzyme activity.

The rat kidney enzyme has the highest reported specific activity. Substrate

specificity studies [26] showed that the enzyme is highly specific for glycine and the

cysteinyl moiety of -glutamylcysteine but not at the -glutamyl moiety. D--glu-

tamyl- and other glutamyl analogs are active using -aminobutyrate in place of

cysteine. This is not likely to be problematic in vivo because the first enzyme,

-glutamylcysteine synthetase is highly specific for L-glutamate [9]. The apparent

Km values for glycine and ATP are 0.37 mM and 34 M, respectively. The apparent

Km determination for -glutamyl--aminobutyrate (a non-thiol analog of -glu-

tamylcysteine) gave non-linear double reciprocal plots that indicated Km apparent

values of 20 or 200 M [26,40]. Further studies on this phenomena are underway

3. Modulation of cellular GSH levels

3.1. GSH deficiency

3.1.1. Inborn errors of glutathione biosynthesis enzymes

Inborn metabolic deficiencies have been described for several GSH-related en-

zymes [4145]. While rare, there are cases of -glutamylcysteine synthetase defi-

ciency. Deficiencies of GSH synthetase occur more frequently than with the first

enzyme. Both -glutamylcysteine synthetase and GSH synthetase deficiencies are

characterized by hemolytic anemia and neurological symptoms. It should be noted

that these are deficiencies not complete absence of enzyme activities. For a more

detailed review, see the chapter by Agne Larsson et al. in this volume.

3.1.2. Experimental GSH deficiency

Although cells from patients with genetic deficiencies of GSH biosynthesis are

available and useful for some studies, the nature of the deficiencies is not yet

understood. GSH metabolism is dynamic, involving many tissues; therefore, in vivo

studies of the effects of GSH deficiency are desirable. Non-specific compounds,

such as diamide, phorone, diethylmaleate, t -butylhydroperoxide, have been used to

reduce GSH levels. Such compounds usually cause non-specific oxidation and other

effects on cells [5]. Thus, it was desirable to develop a specific method for depleting

cellular GSH.

Glutamine is a -glutamyl compound. Methionine sulfoximine (MSO) resembles

glutamine, and it was shown to inhibit glutamine synthetase [46]. Both glutamine


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synthetase and -glutamylcysteine synthetase catalyze the formation of product

via a -glutamyl phosphate intermediate, so it is not surprising that MSO also

inhibits -glutamylcysteine synthetase [47]. Administration of MSO to rodents

leads to convulsions, but it was not known whether inhibition of glutamine

synthetase or of -glutamylcysteine synthetase produces convulsions. Since accep-

tor substrates of the two enzymes are very different (ammonia vs. cysteine), it

was possible to develop selective inhibitors of each enzyme. Glutamine syn-

thetase, but not -glutamylcysteine synthetase, is inhibited by -ethyl methioninesulfoximine (-EtMSO); administration of -EtMSO to rodents leads to de-

creased glutamine levels and produced convulsions [47]. Conversely, -glutamyl-

cysteine synthetase, but not glutamine synthetase, is inhibited prothionine

sulfoximine [15] and more effectively by BSO [16]. Administration of BSO to

rodents leads to dramatically decreased GSH levels (about 10 20% of control

values) with little effect on glutamine levels and there were no convulsions. GSH

is transported out of most cells, so when BSO is administered and GSH synthesis

is inhibited, GSH levels decrease.

Treatment of rodents and/or cells with BSO sensitizes them to the harmful

effects of radiation, melphalan, cyclophosphamide, mercuric ions, cadmium ions

and cisplatin ([5] and references therein). When human lymphocytes and T-cells

are treated with BSO [48], they are no longer able to be activated. Treatment ofneonatal rodents with BSO leads to cataracts [49]. Long term treatment of mice

with BSO produces severe GSH deficiency. Mitochondria do not synthesize GSH,

rather they obtain their GSH from intracellular GSH [50]. While the electron

transport system is highly efficient, some ROS leak. Since mitochondria do not

contain catalase, they depend upon GSH peroxidase and non-enzymatic reaction

with GSH to protect against ROS toxicity. When GSH levels are severely de-

pleted by long term BSO administration, mitochondria swell and cells contain

vacuoles and give a model of endogenous oxidative stress [51,52]. Such mitochon-

drial damage has been observed in skeletal muscle, jejunum, colon and lung type

2 lamellar bodies [51 57]. Such oxidative damage is prevented by GSH esters

(see below) and by ascorbate which appears to spare GSH [5157]. The use of

the BSO model of GSH deficiency has produced some interesting studies that

elucidate some of the functions of GSH and provides a model for test therapies

designed to increase cellular GSH levels.

3.2. Diseases associated with GSH deficiency

A deficiency of GSH occurs in patients with inborn errors of GSH biosynthesis

and in the BSO model of oxidative stress. Low GSH levels have been associated

with the pathology of a number of diseases, such as HIV, hepatitis C, type II

diabetes, ulcerative colitis, burn models, idopathetic pulmonary fibrosis and adult

respiratory distress syndrome (ARDS) and cataracts [3 5,57 59]. GSH is inti-

mately involved in protection against reactive oxygen species (ROS). ROS has been

associated with diseases such as atherosclerosis, ARDS, HIV, rheumatoid arthritis,

cancer, to name a few. GSH deficiency models are useful for understanding the role


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Fig. 3. The structure of 2-oxothiezolidine-4-carboxylic acid.

of GSH in health and disease and for assessing potential therapies for raising

cellular GSH levels.

4. Methods for increasing cellular GSH levels

Increased cellular GSH levels may be beneficial in conditions where GSH levels

are decreased. Administration of cysteine may raise cellular GSH levels because it

is usually the limiting amino acid in GSH biosynthesis [5]. Besides the problems of

oxidation to cystine which has low solubility, cysteine has been reported to be toxic

to cultured cells [60] and to newborn mice [61]. While the mechanism of toxicity isnot known, there are several possibilities [62,63]. Methionine is a precursor of

cysteine through the cystathionase pathway; this pathway may not be active in

newborns and in patients with liver disease. N-acetyl cysteine (NAC) treatment may

increase GSH levels, but it must first be deacetylated to cysteine and both enzymes

for GSH synthesis need to be active. GSH is not taken up by cells to a significant

degree [5]. Administered GSH is degraded extracellularly and the products trans-

ported and used for intracellular GSH biosynthesis. GSH, like methionine and

NAC, is a cysteine delivery agent.

4.1. L-2-oxothiazolidine -4-carboxylic acid

Substrate specificity studies of the -glutamyl cycle enzyme, 5-oxoprolinase

showed that a 5-oxoproline analog, 2-oxothiazolidine-4-carboxylate (OTC), is a

substrate [64] (Fig. 3). The product of the reaction is thought to be S-carboxycys-

teine that is then hydrolyzed to cysteine and CO2. 5-Oxoprolinase is found in many

tissues, with the exception of the lens and erythrocyte. Administration of OTC to

rodents leads to increase tissue cysteine and GSH levels, and such increases are

prevented by treatment with BSO which blocks GSH biosynthesis; thus, OTC is a

cysteine delivery compound [65,66]. OTC protects against acetaminophen toxicity

and is more effective than NAC [66]. When 35S[OTC] was administered to mice,

label was found in all tissues and fluids examined [67]. Radioactive cysteine and

GSH were found in tissues; unmetabolized OTC was found in urine. Administered

OTC increases brain cysteine, but has little effect on brain GSH levels [68]. When

given to sulfur deficient rats, OTC promoted growth and led to increased cellular

GSH levels [69].


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Treatment of cultured human peritoneal mesothelial cells with OTC stimulates

cell proliferation and decreases cell death, suggesting that OTC may be useful in

peritoneal dialysis [70]. OTC treatment protected CHO cells from oxidative stress

[71] and protected isolated rat hearts from ischemic damage [72]. Preliminary

reports [73,74] suggest that OTC treatment decreases AZT induced bone marrow

hypoplasia in mice and augmented the antiviral effects of AZT in cultured cells.

OTC treatment of human lymphocytes is reported [75] to inhibit HIV expression,

HIV-1 promoter activity and NF-KB binding activity in Jurkat cells.Human clinical trials [76] showed that OTC plasma levels peak in about 60 min.

In low dose, plasma cysteine and GSH levels were unaffected over the 8-h study

period. However, lymphocyte cysteine and GSH levels increased 23-fold between

2 and 3 h after OTC administration. In HIV-infected patients [77], treatment with

OTC (100 mg/kg, twice weekly) increased whole blood GSH levels increased over

a 6-week study period and B2-microglobulin levels decreased. Such studies suggest

that further clinical trials are warranted.

4.2. -Glutamylcysteine

Cysteine delivery compounds, such as NAC, GSH, methionine and GSH, may

increase cellular GSH levels, but both enzymes of GSH biosynthesis are needed.Also, the maximum increase is limited by the feedback inhibition of-glutamylcys-

teine synthetase by GSH. Compounds that bypass this feedback inhibited step are

candidates for increasing cellular GSH levels. -Glutamyl amino acids are trans-

ported into kidney and perhaps other tissues. -Glutamylcysteine, its disulfide, and

-glutamylcysteine mixed disulfide with cysteine (-glutamylcystine) are readily

transported into the kidney, reduced, and used by GSH synthetase to form GSH

[78]. When -glutamylcystine was labeled in each cysteine moiety separately and

administered to mice, the cysteine labeled -glutamylcysteine gave rise to a higher

specific activity in GSH than did the labeled cysteine in the mixed disulfide portion

[1,2,5]. These studies support the alternative pathway of GSH biosynthesis dis-

cussed above. Intraventricular administration of-glutamylcysteine led to increased

rat brain GSH levels [79]. Such increases in cellular GSH levels produced after by

-glutamylcysteine administration require that GSH synthetase be active.

4.3. GSH monoester

Since GSH is not significantly taken up by cells [5], an effective approach to

increase cellular GSH levels is to supply a GSH analog that is well transported into

cells and converted into GSH. GSH monoesters, where the ester is in the glycyl

moiety and the ester moiety is ethyl (Fig. 4) or isopropyl, were synthesized [80].

When administered to mice, these GSH monoesters increase cellular GSH levels.

Studies [81] using 35S-labeled GSH monoester showed that it is transported into

many tissues, such as kidney, liver, spleen, lung, heart and red blood cells.

Additional studies showed that GSH levels increase in cerebrospinal fluid [82] and

the lens and brains of newborn rats [83,84]. GSH monoesters have been shown to


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protect even in the presence of BSO, thus showing that GSH monoester does not

have to be degraded and resynthesized into GSH as with GSH itself. GSH ester

treatment protects against toxicity [1,3,5,85] caused by mercuric ions, cadmium

ions, cisplatin, cyclophosphamide, melphalan, radiation, ischemic rat brain damage,

vitamin C deficiency (scurvy) and the BSO model of GSH deficiency. Although

GSH monoester is relatively easy to make, there have been a few reports of toxicity

[85,86]. In the course of our studies, we found that impurities, especially metal ions

may lead to toxicity. We have had no toxicity when copper ions are carefully keptto a minimum [85,87].

4.4. GSH diester

Some of the many preparations of GSH monoester we made, contained GSH

diester (-carboxyl and glycyl) impurities (115%). It was observed that the more

diester impurity, the higher the GSH levels. GSH diester was synthesized [8688]

and was found to be effectively transported into many cells and increased cellular

GSH levels [86,87]. Our studies showed that GSH diester is rapidly transported

into, and out of cells. However, once GSH diester is inside cells, it is rapidly split

into GSH monoester which is more slowly transported than GSH diester. Rodents

are not recommended for studies with the diester because they contain a plasmadiesterase activity that converts GSH diester into GSH monoester. Certain species,

such as humans and hamsters, do not have such a diesterase activity. The

monoester is transported more slowly than the diester, and it is hydrolyzed into

cellular GSH. Preliminary studies suggest that GSH diester raises GSH levels about

four times more effectively than does GSH monoester in hamster liver. In contrast

to GSH monoester, metal ions are not apparently toxic with GSH diester. In our

limited in vivo studies, no toxicity was observed. GSH diester is effective in raising

cellular GSH monoester and GSH levels, and it is a GSH monoester and GSH

delivery compound.

Fig. 4. The structure of gluthathione monoethyl ester; GSH monoethyl ester.


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Acknowledgements

The support by (AI 31804) from the National Institutes of Health and Transcend

Therapeutics is acknowledged.

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