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Glutathione Biosynthesis-Overview PDF
Glutathione Biosynthesis-Overview PDF
Glutathione Biosynthesis-Overview PDF
Abstract
Glutathione (GSH; g-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 g-glutamylcysteine synthetase and GSH synthetase. g-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 g-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; g-Glutamylcysteine synthetase; GSH synthetase; 2-Oxothiazolidine
4-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 [16]. 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: mary@mmcs.memphis.edu
0009-2797/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-2797(97)00146-4
(1)
(2)
The E. coli and rat enzymes are feedback inhibited by GSH [13,14], but not by
its non-thiol analog ophthalmic acid (g-glutamyl-a-aminobutyrylglycine). The apparent 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 g-glutamylcysteine synthetase is probably
not acting at its maximal rate in vivo. Both the rat kidney and the E. coli enzyme
are also inhibited by amino acid sulfoximines that are transition state analogs, such
as 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-g-methylene glutamate [18], S-sulfocysteine and S-sulfohomocysteine [19], Lamino-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 Km value for glutamate returns to about the normal value [24]. While both
the heavy subunit and the holoenzyme are inhibited by GSH, the heavy subunit is
more 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
[22 24]. 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-mercaptoethanol (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.
amino sugar and sialic acid) [26]. There are two asparagine residues and one serine
residue that match the signature for glycosylation. Like g-glutamylcysteine synthetase, rat kidney GSH synthetase shares no significant homology between the E.
coli enzyme. 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 pchloromercuribenzoate 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 that
Cys-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 g-glutamylcysteine but not at the g-glutamyl moiety. D-g-glutamyl- and other glutamyl analogs are active using a-aminobutyrate in place of
cysteine. This is not likely to be problematic in vivo because the first enzyme,
g-glutamylcysteine synthetase is highly specific for L-glutamate [9]. The apparent
Km values for glycine and ATP are 0.37 mM and 34 mM, respectively. The apparent
Km determination for g-glutamyl-a-aminobutyrate (a non-thiol analog of g-glutamylcysteine) gave non-linear double reciprocal plots that indicated Km apparent
values of 20 or 200 mM [26,40]. Further studies on this phenomena are underway
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 is
not 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 transported and used for intracellular GSH biosynthesis. GSH, like methionine and
NAC, is a cysteine delivery agent.
4.2. g-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 g-glutamylcysteine synthetase by GSH. Compounds that bypass this feedback inhibited step are
candidates for increasing cellular GSH levels. g-Glutamyl amino acids are transported into kidney and perhaps other tissues. g-Glutamylcysteine, its disulfide, and
g-glutamylcysteine mixed disulfide with cysteine (g-glutamylcystine) are readily
transported into the kidney, reduced, and used by GSH synthetase to form GSH
[78]. When g-glutamylcystine was labeled in each cysteine moiety separately and
administered to mice, the cysteine labeled g-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 discussed above. Intraventricular administration of g-glutamylcysteine led to increased
rat brain GSH levels [79]. Such increases in cellular GSH levels produced after by
g-glutamylcysteine administration require that GSH synthetase be active.
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 kept
to a minimum [85,87].
10
Acknowledgements
The support by (AI 31804) from the National Institutes of Health and Transcend
Therapeutics is acknowledged.
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14
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